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(12) United States Patent (10) Patent No.: US 8,802,924 B2 Hacohen Et Al

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(12) United States Patent (10) Patent No.: US 8,802,924 B2 Hacohen Et Al USOO8802924B2 (12) United States Patent (10) Patent No.: US 8,802,924 B2 Hacohen et al. (45) Date of Patent: Aug. 12, 2014 (54) POLYUNSATURATED FATTY ACID (2006.01) ELONGASE A638/43 (2006.01) (52) U.S. Cl. (75) Inventors: Zvi Hacohen, Omer (IL); Inna Khozin USPC .......... 800/281:800/295; 435/69.1; 435/134; Goldberg, Sde-Boker (IL); Rivka Ofir, 435/183: 435/320.1; 435/468; 424/94.1; DN Arava (IL); Iskandarov Umidjon, 536/23.7 Sde-Boker (IL) (58) Field of Classification Search (73) Assignee: Ben Gurion University of the Negev None Research and Development Authority, See application file for complete search history. Beer Sheva (IL) (56) References Cited (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S. PATENT DOCUMENTS U.S.C. 154(b) by 437 days. 2005.0089879 A1 4/2005 Feussner et al. .................. 435/6 2005/0214761 A1* 9, 2005 Lerch1 et al. ....... ... 435/6 (21) Appl. No.: 13/132,939 2006/0024404 A1* 2/2006 Kyle ................................. 426.2 (22) PCT Filed: Nov. 26, 2009 OTHER PUBLICATIONS Bigogno et al 2002 Phytochemistry 60: p. 497-503.* (86). PCT No.: PCT/IL2009/001117 Khozin-Goldberg et al 2002 Journal of Phycology 38: p. 991-994.* S371 (c)(1), (2), (4) Date: Aug. 10, 2011 * cited by examiner (87) PCT Pub. No.: WO2010/067352 Primary Examiner — David T Fox Assistant Examiner — Matthew Keogh PCT Pub. Date: Jun. 17, 2010 (74) Attorney, Agent, or Firm — Roach Brown McCarthy & (65) Prior Publication Data Gruber, P.C.; Kevin D. McCarthy US 2011 FO289628A1 Nov. 24, 2011 (57) ABSTRACT An isolated protein which is at least partially encoded by a Related U.S. Application Data polynucleotide sequence encoding a novel elongase is pro (60) Provisional application No. 61/193,589, filed on Dec. vided together with a composition which includes the isolated 9, 2008. protein. A transgenic plant or a transgenic Seed transformed by a polynucleotide encoding a protein which is at least (51) Int. Cl. partially encoded by a novel elongase is also provided. The CI2N 15/87 (2006.01) invention also includes a process for making a very long CI2N 15/82 (2006.01) chain polyunsaturated fatty acid in a transformed cell or plant CI2N IS/00 (2006.01) expressing the isolated protein which is at least partially CI2N 15/74 (2006.01) encoded by a polynucleotide sequence encoding a novel elon CI2N 9/00 (2006.01) gase. CI2P2/06 (2006.01) CI2P 7/64 (2006.01) 20 Claims, 7 Drawing Sheets U.S. Patent Aug. 12, 2014 Sheet 2 of 7 US 8,802,924 B2 O 50 100 150 200 250 3OO Amino acid position Fig. 2 U.S. Patent Aug. 12, 2014 Sheet 3 of 7 US 8,802,924 B2 M. polynorphia ELO I P. paters ELO I M. polynorpha ELO2 P. iFacis ELOI O. tattri ELO L. infantatin ELO2 Thrauzstochytriunza sp. FJN- I O ELO M. alpina ELO I A. Stift ACI O. trif EAO2 T. pseudonana ELO2 C. infestint is ELO/2 X. tropicalis ELO2 O. mykiss R. nor'egicus ELO2 H. sapier as ELO2 A. frcoratziva ELO T. Pseudorzana ELOI Thrausroc tyrrium sp. FJA-10 ELO2 I. galbava ELO9 O. 1 Fig. 3 U.S. Patent Aug. 12, 2014 Sheet 4 of 7 US 8,802,924 B2 A g se 3. is : 3. Y l C t ve d's N . prES2PELOI - 7 pYES2 Retero tie y B sc 3. C. s l pYES2+PiSEO Retention time FIG 4 U.S. Patent Aug. 12, 2014 Sheet 5 Of 7 US 8,802,924 B2 20 16 8 4. O O 3 1 7 14 Time (days) Fig. 5 U.S. Patent Aug. 12, 2014 Sheet 7 of 7 US 8,802,924 B2 Myrmecia incisa) MALTAAWHKYDAIVSRFVFDGLRRVGLOEIOGHPSVITAHLPFIASPTPO 50 Parietochloris incisa) MALTAAWHKYDAIVSRFVFDGLRRVGLQEIQGHPSVITAHLPFIASPTPQ 50 k k k k k k k l r k l k k l k . kirk k r k . (Myrmecia incisa) WTFVLAYLLIVWCGVAALRTRKSSAPREDPAWLRLLVOAHNLVLISLSAY 100 Parietochloris incisa) WTFVLAYLLIVVCGVAALRTRKSSAPREDPAWLRLLVQAHNLVLISLSAY 100 k k k k k k k k k ki ki ki kr k k r rk k k k Ark r k l k k k k k . k k . (Myrmecia incisa) MSSAACYYAWKYGYRFWGTNYSPKERDMGGLIYTYVSKLYEFVDTLIML 15 O Parietochloris incisa MSSAACYYAWKYGYRFWGTNYSPKERDMGGLIYTYVSKLYEFVDTLIML 15 O k k k.k. k.k. k. k.k. k. k.k. k. k. k. k. k. k. k. k. k. k. k. k. k. k. k. k. k.k. k. k. k. k. k. k. k. k.k. k.k. k. k. k. k. k + k k (Myrmecia incisal LKGKVEQVSFLHVYHHASISTIWWAIAYVAPGGDAWYCCFLNSVHVLMY 2 OO Parietochloris incisa LKGKVEQVSFLHVYHHASISTIWWAIAYVAPGGDAWYCCFLNSVHVLMY 2 OO k . k ki ki k . k k ki ki ki krk k (Myrmecia incisa) TYYLLATLLGKDAKARRKYLWWGR LTQFQMFQFVTMMLEAAYTWAYSPY 25 O Parietochloris incisa) TYYLLATLLGKDAKARRKYLWWGRYLTQFQMFQFVTMMLEAAYTWAYSPY 25 O k . k . kirk k (Myrmecia incisal PKFLSKLLFFYMITLLALFANFYAQKHGSSRAAKQKeQ 288 Parietochloris incisa) PKFLSKLLFFYMITLLALFANFYAQKHGSSRAAKQKQ 288 k . k k . k ki ki krk k Fig.7 US 8,802,924 B2 1. 2 POLYUNSATURATED FATTY ACID pathway is further A4 desaturated and finally elongated to ELONGASE docosahexaenoic acid (DHA, 22:603). Fatty acid elongation is a multi-step process involving four sequential enzymatic reactions: rate limiting condensation CROSS-REFERENCE TO RELATED (of malonyl-CoA and acyl-CoA), reduction, dehydration and APPLICATIONS enoyl reduction. Only the expression of the condensing enzyme component is required to reconstitute elongase activ This application is a National Phase Application of PCT ity in the heterologous host; there is no requirement for the International Application No. PCT/IL2009/001117, Interna co-expression of any other component of the elongase com tional Filing Date Nov. 26, 2009, claiming priority of U.S. plex. Multiple microsomal elongation systems with different Provisional Patent Application No. 61/193,589, filed Dec. 9, 10 specificities to the acyl chain length exist in various organ 2008. isms. Recent studies have identified and characterized PUFA specific elongases, responsible for the elongation of PUFA in FIELD OF INVENTION mammals, fish, algae, lower plants and fungi. The elongation of 18:3c)6 to 20:3c)6, the immediate precursor of ARA, was This invention is directed to, interalia, the identification of 15 shown to be the rate limiting step in ARA biosynthesis in M. a cDNA encoding for a P incisa polyunsaturated fatty acid alpina. Functional expression of the PUFA elongase conden elongase (PiELO1) and methods of making and utilizing the sation component in yeast revealed enzymes of various speci SaC. ficities for C18 and C20 acyl substrates. Thus, two types of PUFA elongases engaged in DHA biosynthesis were cloned BACKGROUND OF THE INVENTION from the green microalga Ostreococcus tauri and the diatom Thalassiosira pseudonana: OtBLO1 and TpELO1 are A6 The very long-chain polyunsaturated fatty acid (VLC C18-PUFA specific and involved in the elongation of the PUFA), arachidonic acid (ARA, 20:4(O6), is a component of 18:306 and 18:4c03, while OtBLO2 and TpELO2 are A5 neuron tissues such as brain and retina cells and an important C20-PUFA elongases involved in the elongation of 20:5c)3. component of the human diet. ARA is a primary Substrate for 25 Bifunctional PUFA elongases able to elongate both A6 and A5 the biosynthesis of eicosanoids, including the 2-group pros PUFA, as well as elongases of wide substrate specificity taglandins, 4-group leukotrienes, thromboxanes and lipoxins utilizing both C20 and C22 PUFA substrates, were isolated that serve as biological effectors involved in inflammatory from aquatic animals. and immune responses and cell signaling. Being an important SUMMARY OF THE INVENTION and dominant VLC-PUFA in human breast milk, ARA needs 30 to be externally supplied for normal development of preterm In one embodiment, the present invention provides an iso babies if they are not breast-fed. Due to its beneficial proper lated protein comprising a peptide, wherein the peptide com ties there is a growing interest in the production of ARA for prises an amino acid sequence set forth in SEQID NO: 1. baby formulae. At present, the major commercial source of In another embodiment, the present invention further pro ARA is the filamentous fungus Mortierella alpina. 35 vides a composition comprising an isolated protein compris Microalgae are the most efficient producers and one of the ing a peptide, wherein the peptide comprises an amino acid richest sources of VLC-PUFAs. Furthermore, algae can be sequence set forth in SEQID NO: 1. used as sources of genes for the implementation of VLC In another embodiment, the present invention further pro PUFA biosynthesis in genetically engineered oil crops. The vides an isolated polynucleotide comprising a coding portion genetic information on enzymes involved in the biosynthesis 40 encoding a protein comprising a peptide, wherein the peptide of VLC-PUFA in some algae led to in vivo applications of comprises an amino acid sequence set forth in SEQID NO: 1. VLC-PUFA production in seed oil. The gene pool of the green In another embodiment, the present invention further pro freshwater microalga Parietochloris incisa (Trebouxio vides an isolated polynucleotide comprising a nucleic acid phyceae) is of special interest since it is the only known sequence set forth in SEQID NO: 2. In another embodiment, the present invention further pro microalga able to accumulate extraordinary high amounts of 45 vides a transgenic plant or a transgenic seed transformed by a ARA-rich triacylglycerols (TAG). When P incisa is culti polynucleotide comprising a coding portion encoding a pro vated under nitrogen starvation, the condition triggering Stor tein comprising a peptide, wherein the peptide comprises an age oil accumulation, ARA constitutes about 60 percent of amino acid sequence set forth in SEQID NO: 1. total fatty acids (TFA) and over 95 percent of cellular ARA is In another embodiment, the present invention further pro deposited in TAG in cytoplasmic lipid bodies.
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