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(12) United States Patent (10) Patent No.: US 9,315,837 B2 Umidjon Et Al

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(12) United States Patent (10) Patent No.: US 9,315,837 B2 Umidjon Et Al US009315837B2 (12) United States Patent (10) Patent No.: US 9,315,837 B2 Umidjon et al. (45) Date of Patent: Apr. 19, 2016 (54) DESATURASES OF A GREEN MICROALGA Plant Molecular Biology, Springer, Dordrecht, NL, vol. 54, No. 3, AND USES THEREOF Feb. 1, 2004, pp. 335-352. Domergue Fet al.: "Cloning and functional 1-14 characterization of Phaeodactylum tricornutum from-end desaturases involved in (71) Applicant: Ben Gurion University of the Negev eicosapentaenoic acid biosynthesis'. European Journal of Biochem Research and Development Authority, istry, Published by Springer-Verlag on Behalf of the Federation of Beer Sheva (IL) European Biochemical Societies, vol. 269, No. 16, Aug. 1, 2002, pp. 4015-4113. (72) Inventors: Iskandarov Umidjon, Sde-Boker (IL); Sakuradani E et al.: “Identification of delta 12-fatty acid desaturase from arachidonic acid-producing mortierella fungus by heterologous Inna Khozin Goldberg, Sde-Boker (IL); expression in the yeast Saccharomyces cerevisiae and the fungus Zvi Hacohen, Omer (IL) aspergillusoryzae'. European Journal of Biochemistry, Published by Springer-Verlag on Behalf of the Federation of European Biochemi (73) Assignee: Ben Gurion University of the Negev cal Societies, vol. 261, No. 3, Jan. 1, 1999, pp. 812-820. Research and Development Authority, Los DA et al.: "Structure and expression of fatty acid desaturases”. Beer Sheva (IL) Biochimica Et Biophysica Acta, Elsevier NL, vol. 1394, No. 1, Jan. 1, 1998, pp. 3-15. (*) Notice: Subject to any disclaimer, the term of this Database Geneseq Online Nov. 12, 2009, "Chlorella vulgaris pro patent is extended or adjusted under 35 tein, SEQ ID 4893, accession No. GSP:AWW70226, Database U.S.C. 154(b) by 0 days. accession No. AWW70226. Database Geneseq Online Mar. 20, 2008, “Tetraselmis Suecicadelta (21) Appl. No.: 14/804,638 6 desaturase (TsID6D)-2 enzyme.”..accession No. GSP:AQY10389, Database accession No. AQY10389. Database UniProt Online May 26, 2009, “Micromonas pusilla (22) Filed: Jul. 21, 2015 (strain CCMP1545) (Picoplanktonic green alga).”.accession No. UNIPROT:C1MHO8 Database accession No. CIMHO8. (65) Prior Publication Data Bigogno C et al.: “Lipid and fatty acid compSotion of the green US 2016/0060662 A1 Mar. 3, 2016 oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid”, Phytochemistry, Pergamon Press, GB, vol. 60, No. 5, Jul. 1, 2002, pp. 497-503. Related U.S. Application Data Iskandarov et al. Identification and Characterization of A12, A6, and A5 Desaturases from the Green Microalga Parietochloris incisa, (63) Continuation of application No. 13/520,607, filed as Lipids (2010) 45:519-530. application No. PCT/IL2011/000006 on Jan. 5, 2011, Iskandarov et al. “Identification and Characterization of A12, A6, and now abandoned. A5 Desaturases from the Green Microalga Parietochloris incisa', Lipids (2010) 45: 519-530. (60) Provisional application No. 61/292,185, filed on Jan. Koushirou Suga et al., “Two Low-temperature-inducible Chlorella 5, 2010. Genes for Delta 12 and Iomega-3 Fatty Acid Desaturase (FAD): Isolation of Delta 12 and Iomega-3 fad cDNA Clones, . .”. (51) Int. Cl. Bioscience Biotechnology Biochemistry, vol. 66, No. 6, Jan. 22. CI2N L/20 (2006.01) 2002, pp. 1314-1327, XP055245563, Tokyo, Japan, ISSN: 09 16 CI2P 7/64 (2006.01) 8451, DOI: 10.1271/bbb.66. 1314. CI2N 9/02 (2006.01) Communication and Examination Report from a counterpart foreign CI2N 15/82 (2006.01) application—European Application No. 11731732.1—, mailed Feb. (52) U.S. Cl. 12, 2016; five (5) pages. CPC ............. CI2P 7/6427 (2013.01); CI2N 9/0071 (2013.01); C12N 15/8247 (2013.01); C12Y Primary Examiner — Tekchand Saidha I 14/19 (2013.01) (74) Attorney, Agent, or Firm — Roach Brown McCarthy & (58) Field of Classification Search Gruber, P.C.; Kevin D. McCarthy CPC ......................................................... C12N 1/2O USPC ....................................................... 435/252.3 (57) ABSTRACT See application file for complete search history. Isolated proteins which are at least partially encoded by poly nucleotide sequences encoding novel desaturases are pro (56) References Cited vided together with a composition which includes these iso lated proteins. A transgenic plant, a transgenic alga, or a PUBLICATIONS transgenic seed transformed by the polynucleotides encoding Accession No. D3K944; Submitted Name Delta 12 desaturase proteins which are at least partially encoded by novel desatu (2010). rases are also provided. The invention also includes a process Accession No. D3K945; Submitted Name Delta 6 desaturase (2010). for making a very long-chain polyunsaturated fatty acid in a Accession No. D3K946; Submitted Name Delta 5 desaturase transformed cell, a transgenic alga, or a transgenic plant (Delta-5 fatty acid desaturase) (2010). expressing the isolated protein or proteins which are at least Kajikawa Metal: “Isolation and characterization of A6-desaturase, partially encoded by the polynucleotide sequences encoding and ELO-like enzyme and A5-desaturase from the liverwort novel A5, A6, or A12 desaturases. Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris'. 13 Claims, 16 Drawing Sheets U.S. Patent Apr. 19, 2016 Sheet 2 of 16 US 9,315,837 B2 Z6Z 98Z €6Z O6Z O6Z 68Z ××***|××3x::×*+ U.S. Patent Apr. 19, 2016 Sheet 3 of 16 US 9,315,837 B2 *ON *ON U.S. Patent Apr. 19, 2016 Sheet 4 of 16 US 9,315,837 B2 +x*x?? U.S. Patent Apr. 19, 2016 Sheet 6 of 16 US 9,315,837 B2 U.S. Patent Apr. 19, 2016 Sheet 8 of 16 US 9,315,837 B2 figeodacrispiricaristian m tasissiosira psex&nga — iyagan irregulare k'sigreis giving & Parieicchioris incisa Cercitodon paprelay x desaurrases A-farciania polygorgia rts MattoriciasgadieC. glasia -----. Osiriteras ign:ri 9. Gsteocaecisis facierceritas -Phaeodytan frcoyasis Pihiuin irrgguiare g *istopatistra pegasperata : fictyosteia, cistwice if fortsreia aiia & des: rurases siretaris fas: itar faiei gardii: -:is--------------- parietachiorix3. w i::isa s r $3arciania paixiopia g -issionireiopazers giareiict waiigaris & Parietacitioris icisa €isianxiophoras reirikrikis a dessarisses Spiracia vieraces (sspital his stan fiea &agaeeg Figare 2 U.S. Patent Apr. 19, 2016 Sheet 9 of 16 US 9,315,837 B2 Figure 3A Figure 3B U.S. Patent US 9,315,837 B2 FGURE 4 U.S. Patent Apr. 19, 2016 Sheet 11 of 16 US 9,315,837 B2 {]] CIT ÇIT ÇIT (II {II U.S. Patent Apr. 19, 2016 Sheet 12 of 16 US 9,315,837 B2 U.S. Patent Apr. 19, 2016 Sheet 13 of 16 US 9,315,837 B2 to r . a. s 's st 3. Amino acid position Figars: 6 U.S. Patent Apr. 19, 2016 Sheet 14 of 16 US 9,315,837 B2 M. polymorpha ELO1 P. patenis ELOI M. polymorpha ELO2 P. incis ELO ( tattri ELO L. infantun ELO2 Thraustochytrium sp.F.JN-10 ELOl M. alpirta ELOl P. Saira ELO O. tauri ELO2 T. pseudonana ELO2 C. intestinalis ELOR2 X. tropicalis ELO2 O. mykiss R. norvegicus ELO2 H. sapiens ELO2 tricorriettara ELO T. pseudoriana ELO Thraustochytrium sp. FIN-I0 ELO2 I. gaibara ELO9 O. Figure 7 U.S. Patent Apr. 19, 2016 Sheet 15 of 16 US 9,315,837 B2 r A. g 3. 2 : r Esztigio YES2 Retentors time. Figure 8A s se 8 e s: i. YES Retention tire Figure 88 U.S. Patent Apr. 19, 2016 Sheet 16 of 16 US 9,315,837 B2 8. 3 7 fine (days Figure 9 US 9,315,837 B2 1. 2 DESATURASES OF A GREEN MICROALGA the A6 desaturations, whereas phosphatidylethanolamine AND USES THEREOF (PE) along with PC are the suggested major substrates for the A5 desaturation of 20:3()6 to 20:4()6. The same enzymes are REFERENCE TO CO.-PENDINGAPPLICATIONS involved in the biosynthesis of VLC-PUFA through the co3 pathway in the green microalga Ostreococcus tauri. Priority is claimed as a continuation of U.S. patent appli VLC-PUFAs may also be generated by an alternative A8 cation Ser. No. 13/520,607; filed on Sep. 20, 2012 now aban desaturation pathway. E.g., in the marine haptophyte Isocry doned; which is a 371 of international application number sis galbana and in the fresh water euglenophyte Euglena PCT/IL2011/000006, filed on Jan. 5, 2011; which claims gracilis, where LA and ALA are first elongated by C18 priority to U.S. provisional patent application Ser. No. 10 A9-specific fatty acid elongase followed by sequential A8 and 61/292,185, filed on Jan. 5, 2010. AS desaturations to ARA, DGLA or EPA. The extraplastidial A12 desaturase is an integral ER-bound protein which is FIELD OF INVENTION responsible for the desaturation of oleic acid and production of LA, mainly on phosphatidylcholine (PC). A5 and A6 This invention is directed to, inter alia, proteins having 15 desaturases contain a fused cytochrome b5 domain in their A 12, A6, or A5 desaturase activity, isolated DNA molecules N-terminus, serving as an electron donor, and introduce a encoding the same, and methods of making and utilizing the double bond at a site closer to the carboxyl group than any of SaC. the pre-existing double bonds in the substrate fatty acid, thereby called front-end desaturases. Desaturases with A6 BACKGROUND OF THE INVENTION or A5 activity have been isolated from various organisms, e.g., the nematode C. elegans, the fungus Mortierella alpina, Very long-chain polyunsaturated fatty acids (VLC-PUFA) the moss Physcomitrella patens, the liverwort Marchantia of 20 or 22 carbon atoms are indispensable components of polymorpha and the algae Phaeodactylum tricornutum, human nutrition. They are necessary for normal life-long Thalassiosira pseudonana and Ostreococcus tauri. Some of physiology and benefit the well-being of the human body. 25 these desaturases have been introduced together with PUFA Nutritionally important VLC-PUFAs include the co3-fatty specific elongases into constructs for transformation of yeast acids, eicosapentaenoic acid (EPA, 20:5(O3) and docosa and oil seed plants to reconstitute VLC-PUFA biosynthesis in hexaenoic acid (DHA, 22:603) and the ()6-fatty acid, arachi the heterologous organisms.
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