US 2013 OO19341A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0019341 A1 Umidjon et al. (43) Pub. Date: Jan. 17, 2013 (54) DESATURASES OF A GREEN MICROALGA CI2N I/3 (2006.01) AND USES THEREOF CI2N 5/82 (2006.01) CI2P 7/64 (2006.01) (76) Inventors: Iskandarov Umidjon, Sde-Boker (IL); AOIH 5/00 (2006.01) Inna Khozin Goldberg, Sde-Boker (IL); AOIH 5/10 (2006.01) Zvi Hacohen, Omer (IL) CI2N 15/53 (2006.01) CI2N5/10 (2006.01) (21) Appl. No.: 13/520,607 (52) U.S. Cl. ... 800/281; 435/189: 536/23.2:435/320.1; (22) PCT Filed: Jan. 5, 2011 435/257.2:435/419:435/134; 800/298 e as (86). PCT No.: PCT/IL11AOOOO6 (57) ABSTRACT S371 (c)(1), Isolated proteins which are at least partially encoded by poly (2), (4) Date: Sep. 20, 2012 nucleotide sequences encoding novel desaturases are pro vided together with a composition which includes these iso Related U.S. Application Data lated proteins. A transgenic plant, a transgenic alga, or a (60) Provisional application No. 61/292,185, filed on Jan. transgenic seed transformed by the polynucleotides encoding 5, 2010. proteins which are at least partially encoded by novel desatu rases are also provided. The invention also includes a process Publication Classification for making a very long-chain polyunsaturated fatty acid in a transformed cell, a transgenic alga, or a transgenic plant (51) Int. Cl. expressing the isolated protein or proteins which are at least CI2N 9/02 (2006.01) partially encoded by the polynucleotide sequences encoding CI2N 15/63 (2006.01) novel A5, A6, or A12 desaturases. Patent Application Publication Jan. 17, 2013 Sheet 6 of 16 US 2013/0019341 A1 Patent Application Publication Jan. 17, 2013 Sheet 8 of 16 US 2013/0019341 A1 Phaeodactylum tricornutum Thalassiosira pseudonana Pythium irregulare Mortierella alpina C>Parietochloris incisa 90 Ceratodon purpureus a s gesatrasess 5. Marchantia polymorpha Mantoniella squamata Micromonas pusilla Ostreococcus tauri Ostreococcus lucimarinus Phaeodactylum tricornutum O Pythium irregulare Phytophthora megasperma Dictyostelium discoideum Mortierella alpina S A desatara Ses Ostreococcus tauri Mantoniella squamata {d Parietochloris incisa Marchantia polymorpha Physcomitrella patens S7 Chlorella vulgaris 88 K> Parietochloris incisa Chlamydomonas reinhardtii A desaturases 10 - Spinacia oleracea o Love hirsutum 38 Olea europaea Figure 2 Patent Application Publication Jan. 17, 2013 Sheet 9 of 16 US 2013/0019341 A1 exogenously supplied (asa 2 4 2 Retention time (min) FIGURE 3 Patent Application Publication Jan. 17, 2013 Sheet 10 of 16 US 2013/0019341 A1 100 -- PiDes12 80 -O-PiDess - x - PiDeS5 - -A- - ARA 60 40 35 40 - 30 25 20 O 2 4. 6 8 10 12 14 Time (days) FIGURE 4 Patent Application Publication Jan. 17, 2013 Sheet 13 of 16 US 2013/0019341 A1 o Strict Cutoff OOSe Cutoff ---------- () r h O (9 0. o O op C an W. a on O O 50 1OO 150 2OO 250 3OO Amino acid position Figure 6 Patent Application Publication Jan. 17, 2013 Sheet 14 of 16 US 2013/0019341 A1 M. polymorpha ELO1 P. patens ELOI M. polymorpha ELO2 P. incisa ELOI O, tauri ELOl L. infantum ELO2 Thraustochytrium sp.FJN-10 ELOI M. alpina ELO1 P. salina ELOl O. tauri ELO2 T. pseudonana ELO2 C. intestinalis ELOI/2 X. tropicalis ELO2 O. mykiss R. norvegicus ELO2 H. sapiens ELO2 P. tricornutum ELOI T. pseudonana ELOI Thraustochytrium sp. FJN-10 ELO2 I. galbana ELO9 0.1 Figure 7 Patent Application Publication Jan. 17, 2013 Sheet 15 of 16 US 2013/0019341 A1 2 g (d done 5g Sg so A. e f 3. re s pYES2+PiELO1 Figure 8A B. c. pYES2+PiELO1 - pYES2 Retention tire Figure 8B Patent Application Publication Jan. 17, 2013 Sheet 16 of 16 US 2013/0019341 A1 2O 9 ; 16 O old 12 8 5 4 2 i o O 3 1 7 14 Time (days) Figure 9 US 2013/0019341 A1 Jan. 17, 2013 DESATURASES OF A GREEN MICROALGA Isocrysis galbana and in the fresh water euglenophyte AND USES THEREOF Euglena gracilis, where LA and ALA are first elongated by C18 A9-specific fatty acid elongase followed by sequential FIELD OF INVENTION A8 and AS desaturations to ARA, DGLA or EPA. The extra plastidial A12 desaturase is an integral ER-bound protein 0001. This invention is directed to, interalia, proteins hav which is responsible for the desaturation of oleic acid and ing A2, A6, or A5 desaturase activity, isolated DNA molecules production of LA, mainly on phosphatidylcholine (PC). A5 encoding the same, and methods of making and utilizing the and A6 desaturases contain a fused cytochrome b5 domain in SaC. their N-terminus, serving as an electron donor, and introduce a double bond at a site closer to the carboxyl group than any BACKGROUND OF THE INVENTION of the pre-existing double bonds in the substrate fatty acid, 0002 Very long-chain polyunsaturated fatty acids (VLC thereby called front-end desaturases. Desaturases with A6 PUFA) of 20 or 22 carbon atoms are indispensable compo or A5 activity have been isolated from various organisms, nents of human nutrition. They are necessary for normal e.g., the nematode C. elegans, the fungus Mortierella alpina, life-long physiology and benefit the well-being of the human the moss Physcomitrella patens, the liverwort Marchantia body. Nutritionally important VLC-PUFAs include the polymorpha and the algae Phaeodactylum tricornutum, ()3-fatty acids, eicosapentaenoic acid (EPA, 20:5c)3) and Thalassiosira pseudonana and Ostreococcus tauri. Some of docosahexaenoic acid (DHA, 22:603) and the co6-fatty acid, these desaturases have been introduced together with PUFA arachidonic acid (ARA, 20:4()6) and dihomo-y-linolenic specific elongases into constructs for transformation of yeast acid (DGLA, 20:3(O6) which are the major components of and oil seed plants to reconstitute VLC-PUFA biosynthesis in membrane phospholipids of the retina, brain and testis. ARA the heterologous organisms. and DHA are the predominant fatty acids in the human brain and breast milk. ARA is necessary for normal fetal growth, SUMMARY OF THE INVENTION and cognitive development in infants. Many studies highly Suggested Supplementation of infant formula with DHA and 0006. In one embodiment, the present invention provides ARA. Besides the structural function in membranes, ARA is an isolated protein comprising, an amino acid sequence set the primary Substrate in eicosanoids biosynthesis which regu forth in SEQID NO: 1. lates many physiological processes Such as homeostasis, 0007. In another embodiment, the present invention fur reproduction, immune and inflammatory responses. ther provides an isolated protein comprising, an amino acid 0003 Microalgae are the most efficient producers and one sequence set forth in SEQID NO: 2. of the richest sources of VLC-PUFAs. Furthermore, algae can 0008. In another embodiment, the present invention fur be used as sources of genes for the implementation of VLC ther provides an isolated protein comprising, an amino acid PUFA biosynthesis in genetically engineered oil crops. The sequence set forth in SEQID NO:3. genetic information on enzymes involved in the biosynthesis of VLC-PUFA in some algae led to in vivo applications of 0009. In another embodiment, the present invention fur VLC-PUFA production in seed oil. The gene pool of the green ther provides a composition comprising a protein comprising, freshwater microalga Parietochloris incisa (Trebouxio an amino acid sequence set forth in SEQ ID NO: 1, a com phyceae) is of special interest since it is the only known position comprising a protein comprising, an amino acid microalga able to accumulate extraordinary high amounts of sequence set forth in SEQID NO: 2, a composition compris ARA-rich triacylglycerols (TAG). When P incisa is culti ing a protein comprising, an amino acid sequence set forth in vated under nitrogen starvation, the condition triggering Stor SEQID NO:3, or a composition comprising any combination age oil accumulation, ARA constitutes about 60 percent of thereof. total fatty acids (TFA) and over 95 percent of cellular ARA is 0010. In another embodiment, the present invention fur deposited in TAG in cytoplasmic lipid bodies. ther provides a transgenic plant, a transgenic seed, a trans 0004. The biosynthesis of VLC-PUFA in microalgae fol formed cell, or a transgenic alga transformed by a polynucle lows two major pathways, designated as (D6 and (03. In these otide encoding: (1) a protein comprising, an amino acid pathways, linoleic acid (LA; 18:2()6) and O-linolenic acid sequence set forth in SEQID NO: 1, (2) a protein comprising, (ALA; 18:303) go through sequential. A6 desaturation, A6 an amino acid sequence set forth in SEQ ID NO: 2, (3) a elongation and A5 desaturation, yielding ARA and EPA, protein comprising, an amino acid sequence set forth in SEQ respectively. E.g., in the red microalga Porphyridium cruen ID NO:3, or a transgenic plant, a transgenic seed, or a trans tum and the green microalga P incisa, oleic acid (18:1) is first genic alga transformed by any combination of the polynucle desaturated to LA and Y-linolenic acid (GLA, 18:3(O6) otides (1), (2), and (3). through A12 and A6 desaturations, followed by elongation to 0011. In another embodiment, the present invention fur 20:3 (D6 and A5 desaturation to yield ARA via the ()6 pathway. ther provides a method of producing very long-chain polyun In P incisa, the extraplastidial lipids, phosphatidylcholine saturated fatty acid (VLC-PUFA) in a plant, a plant cell, or an (PC) and the betaine lipid, diacylglyceroltrimethylho alga comprising the step of transforming a plant, an alga, or a moserine (DGTS), are involved in the A12 and, subsequently, plant cell with a polynucleotide encoding: (1) a protein com the A6 desaturations, whereas phosphatidylethanolamine prising, an amino acid sequence set forth in SEQID NO: 1.