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Wo 2010/025374 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 4 March 2010 (04.03.2010) WO 2010/025374 A2 (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12P 7/64 (2006.01) C07C 57/00 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, C12N 1/19 (2006.01) C12N 15/87 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/US2009/055376 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 28 August 2009 (28.08.2009) SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, 61/093,007 29 August 2008 (29.08.2008) US ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, (71) Applicant (for all designated States except US): E. I. DU ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, PONT DE NEMOURS AND COMPANY [US/US]; MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, 1007 Market Street, Wilmington, DE 19898 (US). TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (72) Inventors; and (75) Inventors/Applicants (for US only): HONG, Seung-Pyo Published: [AU/US]; 9 Pierson's Ridge, Hockessin, DE 19707 (US). — without international search report and to be republished SEIP, John, E. [US/US]; P.O. Box 129, Alloway, NJ upon receipt of that report (Rule 48.2(g)) 08001-0129 (US). ZHU, Qun [US/US]; 544 Revere Road, West Chester, PA 19382 (US). — with (an) indication(s) in relation to deposited biological material furnished under Rule I3bis separately from the (74) Agent: CHRISTENBURY, Lynne, M.; E. I. du Pont de description (Rules 13bis.4(d)(i) and 48.2(a)(viii)) Nemours and Company, Legal Patent Records Center, 4417 Lancaster Pike, Wilmington, DE 19805 (US). — with sequence listing part of description (Rule 5.2(a)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: MANIPULATION OF SNFl PROTEIN KINASE ACTIVITY FOR ALTERED OIL CONTENT IN OLEAGINOUS ORGANISMS C-terminus (57) Abstract: Methods of increasing the total lipid content in a eukaryotic cell, the total content of polyunsaturated fatty acids [PUFAs], and/or the ratio of desaturated fatty acids to saturated fatty acids by reducing the activity of the heterotrimeric SNFl protein kinase are disclosed. Preferably, the chromosomal genes encoding the Snfl χ-subunit, Gal83 β-subunit or Snf4 γ-subunit of the SNFl protein kinase, the upstream regulatory genes encoding Sakl, Hxk2, Glkl or Regl, or the downstream genes encod ing Rmel, Cbrl or Snf3 are manipulated in a PUFA-producing strain of the oleaginous yeast Yarrowia lipolytica, resulting in in creased total lipid content, as compared to the parent strain comprising the heterotrimeric SNF 1 protein kinase not having reduced activity. TITLE MANIPULATION OF SNF1 PROTEIN KINASE ACTIVITY FOR ALTERED OIL CONTENT IN OLEAGINOUS ORGANISMS This application claims the benefit of U.S. Provisional Application No. 61/093007, filed August 29, 2008, the disclosure of which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION This invention is in the field of biotechnology. More specifically, this invention pertains to methods useful for manipulating oil content within the lipid fractions of oleaginous organisms based on reduction in activity of the heterotrimehc SNF1 protein kinase, a global regulator of gene expression. BACKGROUND OF THE INVENTION Global regulatory systems modulate the expression of numerous genes located throughout the genome, permitting the overall physiological, metabolic, and developmental status of the organism to respond rapidly and sometimes dramatically to changes in the environment. The term "global regulator" describes a relatively small number of genes whose products have a wide-ranging influence on the state of the cell. One function of these regulators is to code for products that bind promoter elements, such as enhancers or silencers, of the gene whose expression they influence; other regulators function by activating or inactivating a cascading series of cellular reactions. The potential to create powerful regulatory systems in microbial strains using these regulators is only now beginning to be appreciated. The health benefits associated with polyunsaturated fatty acids ["PUFAs'] have been well documented. As a result, considerable research has been directed toward production of large-scale quantities of PUFAs by: 1) cultivation of microbial organisms, such as heterotrophic diatoms Cyclotella sp. and Nitzschia sp.; Pseudomonas, Alteromonas or Shewanella species; filamentous fungi of the genus Pythium; or Mortierella elongata, M. exigua or M. hygrophila, that natively produce the fatty acid of choice; and 2) discovery of fatty acid desaturase and elongase genes that permit synthesis of fatty acids and subsequent introduction of these genes into organisms that do not natively produce ω-3/ω-6 PUFAs via genetic engineering methods. However, commercial exploitation of this work has been limited because of limited production of the preferred ω-3/ω-6 PUFAs and/or inability to substantially improve the yield of oil/control the characteristics of the oil composition produced. Commonly owned U.S. Pat. 7,238,482 describes the use of oleaginous yeast Yarrowia lipolytica as a production host for the production of PUFAs. Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, where greater than 25% of the cellular dry weight is typical. Optimization of the production host has been described in the art (see for example Intl. App. Pub. No. WO 2006/033723, U.S. Pat. Appl. Pub. No. 2006-0094092, U.S. Pat. Appl. Pub. No. 2006-01 15881 , U.S. Pat. Appl. Pub. No. 2006- 0 1 10806 and U.S. Pat. Appl. Pub. No. 2009-0093543-A1 ). The recombinant strains described therein comprise various chimeric genes expressing multiple copies of heterologous desaturases and elongases, and optionally comprise various native desaturase, acyltransferase and peroxisome biogenesis protein knockouts to enable PUFA synthesis and accumulation. Further optimization of the host cell is needed for commercial production of PUFAs. The inventors were interested in identifying a global regulator in oleaginous organisms that would uncouple the process of lipid biosynthesis from the oleaginous stage of growth. Such a regulatory element would be extremely desirable because it would possess broad specificity for the activation and/or repression of secondary metabolite genes while providing strains capable of otherwise normal or near-normal development and growth. It has been found that reduction in activity of the heterothmeric SNF1 protein kinase results in increased accumulation of lipids in Yarrowia lipolytica. Despite numerous previous studies concerning the heterotrimehc SNF1 protein kinase network, many details concerning the cellular role of SNF1 protein kinase and its regulation are still not fully understood and remain to be elucidated. Also, previous studies of SNF1 knockouts have not been performed in an oleaginous organism. Although the AMPK/ SNF1 kinase family is highly conserved throughout eukaryotes and required for the maintenance of cellular energy homeostasis, its specific regulatory mode may be different among different organisms. This appears to be the first discovery of a generalized mechanism wherein reduction in the activity of the heterotrimeric SNF1 protein kinase leads to the surprising discovery of increased lipid biosynthesis, resulting in constitutive oleaginy. SUMMARY OF THE INVENTION In a first embodiment, the instant invention concerns a transgenic oleaginous eukaryotic host cell comprising: (a) a heterotrimeric SNF1 protein kinase having reduced activity when compared to the activity of a heterotrimeric SNF1 protein kinase of a non-transgenic oleaginous eukaryotic host cell; and, (b) an increase in total lipid content when compared to the total lipid content of a non-transgenic oleaginous eukaryotic host cell comprising a heterotrimeric SNF1 protein kinase not having reduced activity. In a second embodiment, the invention concerns the transgenic oleaginous eukaryotic host cell as described herein wherein the reduction in activity of the heterotrimeric SNF1 protein kinase and increase in total lipid content is due to a modification selected from the group consisting of: (a) altering an upstream regulatory protein associated with the heterotrimeric SNF1 protein kinase; (b) altering a polynucleotide encoding a subunit of the heterotrimeric SNF1 protein kinase; and, (c) altering a downstream protein regulated by the heterotrimeric SNF1 protein kinase. In a third embodiment, the invention concerns a transgenic oleaginous eukaryotic host cell of of the invention wherein the reduction in activity of the heterotrimeric SNF1 protein kinase and increase in total lipid content is due to a modification selected from the group consisting
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