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

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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. donic acid (ARA, 20:4(O6) and dihomo-y-linolenic acid (DGLA, 20:3c)6) which are the major components of mem 30 SUMMARY OF THE INVENTION brane phospholipids of the retina, brain and testis. ARA and DHA are the predominant fatty acids in the human brain and In one embodiment, the present invention provides an iso breast milk. ARA is necessary for normal fetal growth, and lated protein comprising, an amino acid sequence set forth in cognitive development in infants. Many studies highly Sug SEQID NO: 1. gested supplementation of infant formula with DHA and 35 In another embodiment, the present invention further pro ARA. Besides the structural function in membranes, ARA is vides an isolated protein comprising, an amino acid sequence the primary Substrate in eicosanoids biosynthesis which regu set forth in SEQID NO: 2. lates many physiological processes Such as homeostasis, In another embodiment, the present invention further pro reproduction, immune and inflammatory responses. vides an isolated protein comprising, an amino acid sequence Microalgae are the most efficient producers and one of the 40 set forth in SEQID NO:3. richest sources of VLC-PUFAs. Furthermore, algae can be In another embodiment, the present invention further pro used as sources of genes for the implementation of VLC vides a composition comprising a protein comprising, an PUFA biosynthesis in genetically engineered oil crops. The amino acid sequence set forth in SEQID NO: 1, a composi genetic information on enzymes involved in the biosynthesis tion comprising a protein comprising, an amino acid of VLC-PUFA in some algae led to in vivo applications of 45 sequence set forth in SEQID NO: 2, a composition compris VLC-PUFA production in seed oil. The gene pool of the green ing a protein comprising, an amino acid sequence set forth in freshwater microalga Parietochloris incisa (Trebouxio SEQID NO:3, or a composition comprising any combination phyceae) is of special interest since it is the only known thereof. microalga able to accumulate extraordinary high amounts of In another embodiment, the present invention further pro ARA-rich triacylglycerols (TAG). When P incisa is culti 50 vides a transgenic plant, a transgenic seed, a transformed cell, vated under nitrogen starvation, the condition triggering Stor or a transgenic alga transformed by a polynucleotide encod age oil accumulation, ARA constitutes about 60 percent of ing: (1) a protein comprising, an amino acid sequence set total fatty acids (TFA) and over 95 percent of cellular ARA is forth in SEQ ID NO: 1, (2) a protein comprising, an amino deposited in TAG in cytoplasmic lipid bodies. acid sequence set forth in SEQID NO: 2, (3) a protein com The biosynthesis of VLC-PUFA in microalgae follows two 55 prising, an amino acid sequence set forth in SEQID NO:3, or major pathways, designated as ()6 and (03. In these pathways, a transgenic plant, a transgenic seed, or a transgenic alga linoleic acid (LA; 18:2006) and O-linolenic acid (ALA; transformed by any combination of the polynucleotides (1), 18:3(O3) go through sequential. A6 desaturation, A6 elonga (2), and (3). tion and A5 desaturation, yielding ARA and EPA, respec In another embodiment, the present invention further pro tively. E.g., in the red microalga Porphyridium cruentum and 60 vides a method of producing very long-chain polyunsaturated the green microalga P incisa, oleic acid (18:1) is first desatu fatty acid (VLC-PUFA) in a plant, a plant cell, or an alga rated to LA and gamma.-linolenic acid (GLA, 18:3()6) comprising the step of transforming a plant, an alga, or a plant through A12 and A6 desaturations, followed by elongation to cell with a polynucleotide encoding: (1) a protein comprising, 20:3 (D6 and A5 desaturation to yield ARA via the ()6 pathway. an amino acid sequence set forth in SEQ ID NO: 1, (2) a In P incisa, the extraplastidial lipids, phosphatidylcholine 65 protein comprising, an amino acid sequence set forth in SEQ (PC) and the betaine lipid, diacylglyceroltrimethylho ID NO: 2, (3) a protein comprising, an amino acid sequence moserine (DGTS), are involved in the A12 and, subsequently, set forth in SEQID NO:3, or transforming a plant, a plant US 9,315,837 B2 3 4 cell, or an alga with any combination of the polynucleotides ment was generated by the CLUSTALW program and the (1), (2), and (3), thereby producing a VLC-PUFA in a plant, a unrooted phylogram was constructed by the neighbor-joining plant cell, or an alga. method using the MEGA4 software. GeneBank accession numbers for the PUFA elongases are: ACK99719 (A6, P BRIEF DESCRIPTION OF THE DRAWINGS incisa), AAV67797 (A6, O. tauri), AAV67798 (A5, O. tauri), AAT85662 (A6, M. polymorpha), BAE71129 (A5, M. poly FIGS. 1A through 1G depict the deduced amino acid morpha), AAL84174 (A6, P. patens), CAJ 30819 (A6, sequences of P incisa PiDes 12 (A), PiDesG (B), and PiDes5 Thraustochytrium sp.), CAM55873 (A5, Thraustochytrium (C) are aligned with their closest homologs using CLUSTAL sp.), AAF70417 (A6, M. alpina), XP 001467802 (L. infan W (1.83) multiple sequence alignment program (default). 10 Conserved motifs characteristic of each desaturase sequence tum), AAV67803 (A6/A5, O. mykiss), NP_001029014 (A6/ are highlighted. GeneBank accession numbers for the A5, C. intestinalis), NP 068586 (A6/A5, H. sapiens), sequences are: A) C. reinhardtii (XP 001691669); C. vul AAY15135 (A5, P. salina), CAM55851 (A6P tricornutum), garis (BAB78716), G. hirsutum (AAL37484), S. Oleracea AAL37626 (A9, I. galbana), AAV67799 (A6, T. pseud (BAC22091), O. europaea (AAW63040). B) M. polymorpha 15 onana), AAV67800 (A5, T. pseudonana), CAA92958 (A6, C. (AAT85661), P tricornutum (AAL92563), T. pseudonana elegans), NP 599209 (A6/A5, R. norvegicus). (AAX 14505), O. tauri (AAW70159), M. squamata FIGS. 8A and 8B area GC plot of FAMES of recombinant (CAQ30479). C) O. tauri (CAL57370), M. squamata yeast harboring pYES2 and PiELO1 fed with 18:306 (A) and (CAQ30478), M. polymorpha (AAT85663), D. discoideum 18:403 (B) CONTROL. (BAA37090), M. alpina (AAC72755), P tricornutum FIG. 9 is a bar graph Summarizing the results of quantita (AY082392); and FIGS. 1A-1G are collectively referred to as tive Real-time RT-PCR analysis of PiELO1 gene expression just FIG. 1. in log phase (Time 0) and N-starved (3, 7 & 14 d) cells of P FIG. 2 is an unrooted phylogram of PiDes 12, PiDesG. incisa. The transcript abundance of the gene was normalized PiDes5 and some functionally characterized A12, A6 and A5 to 18S SSU rRNA gene. desaturases (vertebrate and invertebrate desaturases are not 25 included). The alignment was generated by the CLUSTALW program and the unrooted phylogram was constructed in the DETAILED DESCRIPTION OF THE INVENTION neighbor-joining method using the MEGA4 software 47. GeneBank sources of the sequences are: BAB78716 (A12, In one embodiment, the present invention provides an iso Chlorella vulgaris), XP 001691669 (A12, C. reinhardtii), 30 lated protein. In another embodiment, the present invention BAC22091 (A12, Spinacia oleracea), AAL37484 (A12, Gos provides that the isolated protein is a polypeptide. In another sypium hirsutum), AAW63040 (A12, Olea europaea), embodiment, the present invention provides that the isolated CAB94993 (A6, Ceratodon purpureus), AAT85661 (A6, M. protein is an enzyme. In another embodiment, the present polymorpha), BAA85588 (A6, M. alpina), AAL92563 (A6, P invention provides that the isolated protein is a desaturase. In tricornutum), AAX 14505 (A6, T. pseudonana), (A6, Pythium 35 another embodiment, the present invention provides that the irregulare), CAL57370 (A5, O. tauri), AAT85663 (A5, M. isolated protein is an algal desaturase. In another embodi polymorpha), AAL13311 (A5, P irregulare), CAD53323 ment, the present invention provides that the isolated protein (A5, Phytophthora megasperma), BAA37090 (A5, Dictyoste is a microalgae desaturase. In another embodiment, the lium discoideum), AAC72755 (A5, M. alpina), CAQ30478 present invention provides that the isolated protein is a A12 (A5, M. squamata), CAQ30479 (A6, M. squamata), 40 desaturase. In another embodiment, the present invention AAW70159 (M. O. tauri), CS020055 (A5, P. patens). provides that the isolated protein is a A6 desaturase. In FIGS. 3A and B provide graphs representing GC FAMES another embodiment, the present invention provides that the of recombinant yeast harboring pYES2 (control), pY PiDesG isolated protein is a A5 desaturase. In another embodiment, (A) fed with 18:2 or 18:303, and pYPiDes5 (B) fed with the present invention provides that the isolated protein is a 20:3 (D6, 20:4c)3 or 18:1; and those graphs are collectively 45 microalgae desaturase produced in a plant cell. In another referred to as just FIG. 3. embodiment, the present invention provides that the isolated FIG. 4 is a graph showing the changes in expression of the protein is a microalgae desaturase produced in an algal cell. PiDes 12, PiDesG, and PiDes5 genes under N-starvation and In another embodiment, the present invention provides a ARA percent share in total fatty acids. The transcript abun A12 desaturase comprising the amino acid sequence: dance of the genes was normalized to that of the actin gene. 50 FIGS.5A and B depict the amino acid sequence of P incisa PiELO1 aligned with its closest homologs using CLUSTAL (SEQ ID NO: 1) W (1.83) multiple sequence alignment program (default). MGKGGCYOAGPPSAKKWESRVPTAKPEFTIGTLRKAIPVHCFERSIPRSF Conserved motifs characteristic of PUFA elongase sequences AYLAADLAAIAWMYYLSTFIDHPAVPRVLAWGLLWPAYWYFOGAVATGVW are highlighted. GeneBank accession numbers for the 55 sequences are OtELO1 (O. tauri, AAV67797), MpELO1 (M. VIAHECGHQAFSPYOWLNDAVGLVLHSCLLVPYYSWKHSHRRHHSNTGST polymorpha, AAT85662), PpELO1 (P. patens, AAL84174), MpELO2 (M. polymorpha, BAE71129), and ThrELO1 TKDEVFWPREAAMVESDFSLMOTAPARFLVIFWSLTAGWPAYLFANASGR Thraustochytrium sp. FIN-10, ABC18313); since FIGS. 5A KYGKWANHFDPYSPIFTKRERSEIWWSDWALTWWIAGLYSLGKAFGWAWL and 5B are the same figure, they will be collectively referred 60 to as FIG. 5. WKEYVIPYLIVNMWLVMITLLOHTHPELPHYADKEWDWLRGALATCDRSY FIG. 6 is a hydropathy plot of the amino acid sequence of PiELO1. The lower dashed line and the upper line represent GGMPDHLHHHIADTHVAHHLFSTMPHYHAOEATEAIKPILGKYYKODKRN the loose transmembrane region cutoff and the strict trans WWAALWEDFSLCRYWAPDTAGSGILWFRA membrane region cutoff, respectively. 65 FIG. 7 is an unrooted phylogram of PiELO1 and some In another embodiment, the present invention provides a other functionally characterized PUFA elongases. The align A6 desaturase comprising the amino acid sequence: US 9,315,837 B2 6 60% identical to the amino acid sequence of SEQID NO: 1. (SEQ ID NO: 2) SEQID NO:2, or SEQID NO:3. In another embodiment, the MCOGOAWOGLRRRSSFLKLTGDAIKGAVAAISDFNKLPAATPWFARRSLS desaturase comprises an amino acid sequence that is at least DSALOORDGPRSKOOVTLEELAOHNTPEDCWLVIKNKVYDVSGWGPOHPG 70% identical to the amino acid sequence of SEQID NO: 1. SEQID NO:2, or SEQID NO:3. In another embodiment, the GHVIYTYAGKDATDVFACFHAOTTWSOLRPFCIGDIVEEEPMPALLKDFR desaturase comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQID NO: 1. ELRTRLOOOGLFRSNKLYYLYKVASTLSLLAAALAVLITORDSWLGLVGG SEQID NO:2, or SEQID NO:3. In another embodiment, the AFLLGLFWOOSGWLAHDFLHHOVFTDROWNNWMGYFLGNVCOGFSTDWWK desaturase comprises an amino acid sequence that is at least 10 80% identical to the amino acid sequence of SEQID NO: 1. SKHNWHHAWPNELDSDSKAARDPDIDTLPLLAWSSEMLDSMSNSGARLFW SEQID NO:2, or SEQID NO:3. In another embodiment, the RMOHYFFFPILLFARMSWCOOSVAHASDLSRTSKAGVYELAYLALHYAWF desaturase comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQID NO: 1. LGAAFSVLPPLKAVWFALLSOMFSGFLLSIVFVOSHNGMEWYSDTKDFWT SEQID NO:2, or SEQID NO:3. In another embodiment, the 15 desaturase comprises an amino acid sequence that is at least AOIVSTRDILSNVWNDWFTGGLNYOIEHHLFPTLPRHNLGKVOKSIMELC 90% identical to the amino acid sequence of SEQID NO: 1. HKHGLVYENCGMATGTYRVLORLANVAAEA SEQID NO:2, or SEQID NO:3. In another embodiment, the desaturase comprises an amino acid sequence that is at least In another embodiment, the present invention provides a 95% identical to the amino acid sequence of SEQID NO: 1. AS desaturase comprising the amino acid sequence: SEQID NO:2, or SEQID NO:3. In another embodiment, the desaturase comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQID NO: 1. (SEQ ID NO : 3) SEQID NO: 2, or SEQID NO: 3. MMAVTEGAGGVTAEVGLHKRSSOPRPAAPRSKLFTLDEVAKHDSPTDCWV In another embodiment, the desaturase as described herein VIRRRVYDWTAWVPOHPGGNLIFVKAGRDCTOLFDSYHPLSARAVLDKFY 25 comprises at least a portion of the amino acid shown in SEQ ID, NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In another IGEVDVRPGDEOFLVAFEEDTEEGOFYTVLKKRVEKYFRENKLNPRATGA embodiment, the desaturase as described herein is a variant of MYAKSLTILAGLALSFYGTFFAFSSAPASLLSAWLIGICMAEWGWSIMHD SEQID. NO: 1, SEQID NO: 2, or SEQID NO:3. In another embodiment, the term “variant' in relation to a certain ANHGAFARNTWASHALGATLDIVGASSFMWRQOHV VGHHAYTNVDGODPD 30 sequence means a protein or a polypeptide which is derived from the sequence through the insertion or deletion of one or LRVKDPDWRRVTKFQPQOSYQAYOHIYLAFLYGLLAIKSWLLDDFMALSS more amino acid residues or the substitution of one or more GAIGSWKWAKLTPGEKLWFWGGKALWLGYFWLLPWWKSRHSWPLLAACWL amino acid residues with amino acid residues having similar properties, e.g., the replacement of a polar amino acid residue LSEFWTGWMLAFMFOVAHVTSDVSYLEADKTGKVPRGWAAACAATTADFA 35 with another polar amino acid residue, or the replacement of HGSWFWTOISGGLNYOVWHHLFPGICHLHYPAIAPIVLDTCKEFNVPYHV a non-polar amino acid residue with another non-polar amino acid residue. In all cases, variants must have a desaturase YPTFWRALAAHFKHLKDMGAPTAIPSLATWG function as defined herein. In another embodiment, the desaturase of the present In another embodiment, the desaturase as described herein 40 further comprises a leader peptide. In another embodiment, invention comprises an amino acid sequence that is at least the leader peptide allows the polypeptide to be specifically 60% homologous to the amino acid sequence of SEQID NO: located or targeted to a target organelle within the cell. In 1, SEQID NO: 2, or SEQID NO:3. In another embodiment, another embodiment, the desaturase as described herein fur the desaturase comprises an amino acid sequence that is at ther comprises a sequence motif responsible for microsomal least 70% homologous to the amino acid sequence of SEQID 45 localization. In another embodiment, a desaturase as NO: 1, SEQNO:2, or SEQID NO:3. In anotherembodiment, described herein further comprises chemical modification the desaturase comprises an amino acid sequence that is at Such as glycosylation that increases its stability. In another least 75% homologous to the amino acid sequence of SEQID embodiment, a desaturase as described herein further com NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi prises a peptide unrelated to desaturase which increases its ment, the desaturase comprises an amino acid sequence that is 50 stability. at least 80% homologous to the amino acid sequence of SEQ In another embodiment, the present invention provides an ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In another isolated PUFA desaturase. In another embodiment, the embodiment, the desaturase comprises an amino acid present invention provides an isolated polypeptide compris sequence that is at least 85% homologous to the amino acid ing a functional long chain polyunsaturated fatty acid (PUFA) sequence of SEQID NO: 1, SEQID NO: 2, or SEQID NO: 55 desaturase. In another embodiment, the present invention 3. In another embodiment, the desaturase comprises an amino provides that the polypeptide has the function of desaturating acid sequence that is at least 90% homologous to the amino a chain longer than 18 carbons fatty acid. In another embodi acid sequence of SEQID NO: 1, SEQID NO: 2, or SEQID ment, the present invention provides that the polypeptide has NO:3. In another embodiment, the desaturase comprises an the function of desaturating a chain longer than 20 carbons amino acid sequence that is at least 95% homologous to the 60 fatty acid. amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or In another embodiment, the present invention provides an SEQID NO:3. In another embodiment, the desaturase com isolated PUFA desaturase comprising a fused N-terminal prises an amino acid sequence that is at least 98% homolo cytochrome b5 domain. In another embodiment, the present gous to the amino acid sequence of SEQID NO: 1, SEQID invention provides an isolated PUFA desaturase which NO: 2, or SEQ ID NO:3. 65 desaturates (06 substrates. In another embodiment, the In another embodiment, the desaturase of the present present invention provides an isolated PUFA desaturase invention comprises an amino acid sequence that is at least which desaturates both (O3 substrates. In another embodi US 9,315,837 B2 7 8 ment, the present invention provides an isolated PUFA otide comprises a DNA sequence encoding a polypeptide desaturase which desaturates both (03 and (06 substrates. In consisting a desaturase activity. another embodiment, the present invention provides an iso In another embodiment, the isolated polynucleotide com lated PUFA desaturase encoded by SEQID NO: 1 (PiDes 12 prises a DNA sequence comprising the sequence: or A12). In another embodiment, the present invention pro vides an isolated PUFA desaturase encoded by SEQID NO: 2 (PiDesG or A6). In another embodiment, the present inven (SEQ ID NO: 4, PiDes12) tion provides an isolated PUFA desaturase encoded by SEQ atggggaaaggaggctgttaccaggc.cgggcctic ctago.gcaaagaaatg ID NO: 3 (PiDes5 or A5). In another embodiment, the sub ggagagtagggtgcc cactgccaaac cc.gagttcacgat.cggalaccCtcC strate for the present invention isolated PUFA desaturase is 10 18:2006, 20:306, 20:4c03, and 20:303 gcaaagctat accogg to cactgctt.cgaacggtc. catcc ct cqgt cattg In another embodiment, the present invention provides an Cctaccttgcggcagacctggcggct attgcggt catgtactacctgagc isolated PUFA desaturase which desaturates 20:303 to a non actitt catcgatcatc.ccgc.cgtgcc.gcgggtc.ctggcctggggtttgct methylene-interrupted 20:4. In another embodiment, the 15 present invention provides that PiDes5 desaturates 20:3c)3 to gtggcCtgcc tactggtact tccalaggtgctgtggcgacaggcgtctggg a non-methylene-interrupted 20:4. In another embodiment, the present invention provides that PiDes5 converts 20:4c)3 tgattgct cacgagtgcggccaccaggcgttcticgc.cctaccagtggctic into the respective A5 product, 20:5c)3 (EPA) as well as the aacgacgctgtggggcttgttgctgcact Cotgcttgctggtgc cct atta added 18:1 into the non-methylene-interrupted 18:2^. In another embodiment, the present invention provides a ct cotggaag cact cacacagacggcaccact coaacaccggaagicacca protein comprising a desaturase activity. In another embodi ccaaggatgaggtgtttgtc.ccc.cgggalagcagc catggtggagt cqgac ment, the present invention provides a protein consisting a desaturase activity. In another embodiment, the present ttct cottgatgcagacagct cocqcgcggttcc tiggtcatctitcgt.ctic invention provides that the protein of the invention is a recom 25 gctgaccgctggctggcctgcctacctgtttgccaatgcatctggcc.gca binant desaturase. In another embodiment, the present inven tion provides that the desaturase is a polyunsaturated fatty agtatggcaa.gtgggccaaccactittgacc cc tact cacccatctt cacc acid (PUFA)-specific desaturase. In another embodiment, the present invention provides that the desaturase desaturates aa.gc.gc.gagcgcago.gagat.cgttgtcagcgatgtc.gc.gctgacggtggit precursors of arachidonic acid. In another embodiment, the 30 catcgcggggctic tact cqctgggcaaggcgtttggctgggcctggctgg present invention provides that the desaturase desaturates precursors of EPA. In another embodiment, the present inven tdaaggagtatgtgat CCCCtacct catcgtcaiacatgtggctggtcatg tion provides that the desaturase desaturates immediate pre at Cacgctgctgcagcacacgcaccc.cgagctg.ccgcactacgc.cgacaa cursors of arachidonic acid (ARA). In another embodiment, the present invention provides that the protein as described 35 ggagtgggactggctg.cgcggcgc.gctggccacctg.cgatcgcagctacg herein is used to elevate PUFA levels in animals, thereby providing a ready source of PUFAs. gcgg catgc.cggaccacctgcaccacca catcgc.cgacacgcacgtc.gct In another embodiment, the expression and/or transcrip Caccacctgttct coac catgcc.gcactaccatgcgcaggaggcgactga tion of the desaturase as described herein is up-regulated during nitrogen starvation. In another embodiment, the 40 ggcgat Caagcc catcctgggcaagtact acaag Cagga caag.cgcaacg expression and/or transcription of the desaturase as described herein is up-regulated under oleogenic conditions. In another totgggcagcgct Ctgggaggattt Cagcctgtgcc.gctatgtggcgc.ct embodiment, oleogenic conditions comprise the presence of gacacagcaggct cqggcatcCtgtggttcc.gc.gcttga a A6 substrate for A6 or A5 fatty acid desaturase. In another embodiment, oleogenic conditions comprise 18:2006 and 45 In another embodiment, SEQ ID NO. 4 encodes the amino 20:3(O6. In another embodiment, oleogenic conditions com acid sequence of SEQID NO: 1. prise nitrogen starvation. In another embodiment, the expres In another embodiment, the isolated polynucleotide com sion and/or transcription level of the desaturases as described prises a DNA sequence comprising the sequence: herein correlates with the production of ARA precursors. In another embodiment, oleogenic conditions comprise nitro 50 gen starvation. In another embodiment, the expression and/or (SEQ ID NO : 5, PiDesé) transcription level of the desaturases as described herein cor atgtgcCagggacaggcagtic cagggtctgaggcgc.cggagttcatt atg relates with the production of DGLA precursors, EPA pre aagctic accggggacgctatcaaagggg.ccgttgcc.gcaat at Cagacitt cursors, DHA precursors, ARA precursors, or any combina tion thereof. 55 caacaa.gctic ccggcc.gcaacgc.cagtgttcgc.caggcggit cactitt cog In another embodiment, the present invention provides an isolated polynucleotide encoding the protein as described acagcgctctgcago agcgagatggc.ccgc.gcagcaa.gcagcagg to acc herein. In another embodiment, an isolated polynucleotide is Ctggaagagctagogcagcataatacgc.ctgaggattgctggctggt cat an isolated DNA molecule. In another embodiment, an iso Caagaacaaggtgtacgacgt cagcggttggggaccgcago accc.cggtg lated polynucleotide is an isolated cDNA molecule. In 60 another embodiment, the isolated polynucleotide comprises a ggcacgtgat ctatacgitatgctggcaaagacgc.cacggacgtttittgcc sequence encoding the protein as described herein. In another embodiment, the isolated polynucleotide comprises a DNA tgat coatgcc.ca.gaccacttggtc.gcagttgaga.ccct tctgcatcggg sequence encoding a desaturase as described herein. In gacattgttggaggaggagccalatgc.cggcgctgctcaaagact tcc.gcga another embodiment, the isolated polynucleotide comprises a 65 DNA sequence encoding a polypeptide comprising a desatu gctg.cgcacccggctgcagcagcagggcctgttt cqcagcaacaa.gagta rase activity. In another embodiment, the isolated polynucle US 9,315,837 B2 9 10 - Continued - Continued Ctacagtaca aggtggc.ca.gcacgctgagcct actggcggcc.gc.gctggc Ctggcc at Caagagcgtgctgctggacgactittatggcc ct cagctc.cgg agtgctgat cacgcagcgcgact cotggctgggit ct cqtcggcggcgcgt. cgc.cat cqgct cogtgaaagtggccaagctgacgc.ccggcgagaa.gctcg t cct gctgggcct Cttaggcagcagtc.gggctggctggcgcacgact tcc tgttctgggg.cggcaaggcgctctggct cqgct actttgttgctgctg.ccg tgcaccaccaggtott Caccgaccgc.cagtggaacaacgtgatgggctac gtggtgaagagcc.gc.cact Cotggcc.gctgctggcggcctgctggctgct titcCtgggcaacgtctgcc agggat cagdacggactggtggalaga.gcaa.g gagggagtttgtcacgggctggatgctggcct tcatgttcCaggtggcgc

Cacaacgtgcaccacgcggtgcc caacgagct cacagcgacagcaaggc 10 acgtgaccagcgatgtgagctacctggaggctgacaaga Caggcaaggt C ggcgcgggaccc.cgacatcgacacgctgcc cctgctggcctggagct cqg cc gaggggctgggctgcc.gcacaggcc.gc.caccaccgc.cgactittgcgca agatgctgga cago atgagcaacticgggcgc.gc.gc.ctgtttgttgcgcatg tggctic ctggttctggacc caaatttctggcggcct taactaccaggtgg cago act acttcttct tcc ccatcc togct citt cqcgc.gcatgtcc tdgtg 15 tgcaccatctgttc.ccgggcatctgc catctgcact acccggc catcgc.c Ccagcagtctgtc.gc.gcacgc.ct cqgacctgtcCaggacct caaaggcgg Ccatcgtgctgga cacctgcaaggagtttalacgtgc cctaccatgtgtac gcgtgt atgagctggcgitaticttgcgctgcattatgcctggttcCtgggc cc cacgtttgtcagggcactic go cqcacacttcaag catct caagga cat gcggcCtt cagcgtgct cocgc.ccct Caaggcggtcgtgttcgc.gctgct gggcgc.cccaactgc catcCCtt.cgctggccaccgtgggatag cago cagatgttitt coggct tcc togct ct c catcgt.ctttgtgcagagcc In another embodiment, SEQ ID NO: 6 encodes the amino acid sequence of SEQID NO:3. acaacggcatggaggtgtacagcgacacaaaggactttgttgacggcc cag In another embodiment, the isolated polynucleotide com attgttgtc. cacgc.gcga catattgtcaaacgtctggaacgactggttcac prises a DNA sequence that is at least 60% homologous to the 25 nucleic acid sequence of SEQID NO:4, SEQID NO: 5, SEQ aggcgggctgaactaccagat.cgag caccacctgttc.cccacgctg.ccgc ID NO: 6, or SEQ ID NO: 8. In another embodiment, the isolated polynucleotide comprises a DNA sequence that is at gccaca acctgggcaaggit coagaagt ccatcatggagctgtgccacaag least 70% homologous to the nucleic acid sequence of SEQ Catggcctggtgtacgaaaactg.cggcatggctactggcacct atctgt ID NO:4, SEQID NO:5, SEQID NO: 6, or SEQID NO:8. 30 In another embodiment, the isolated polynucleotide com gctgcagcgc.ctggcaaacgtggcagctgaggcc tag prises a DNA sequence that is at least 75% homologous to the nucleic acid sequence of SEQID NO:4, SEQID NO: 5, SEQ In another embodiment. SEQ ID NO; 5 encodes the amino acid sequence of SEQID NO: 2. ID NO: 6, or SEQ ID NO: 8. In another embodiment, the 35 isolated polynucleotide comprises a DNA sequence that is at In another embodiment, the isolated polynucleotide com least 80% homologous to the nucleic acid sequence of SEQ prises a DNA sequence comprising the sequence: ID NO:4, SEQID NO:5, SEQID NO: 6, or SEQID NO:8. In another embodiment, the isolated polynucleotide com prises a DNA sequence that is at least 85% homologous to the (SEQ ID NO: 6, PiDess) atgatggctgta acagagggcgctgggggtgtaacggc.cgaggttggttt 40 nucleic acid sequence of SEQID NO:4, SEQID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In another embodiment, the gcacaaacgcagttct cagcc.gcgt.ccc.gcagct coccgcagdaagctgt isolated polynucleotide comprises a DNA sequence that is at least 90% homologous to the nucleic acid sequence of SEQ t cacgttggatgaggttgcaaag cacgacagc.ccgactgactgctgggtg ID NO:4, SEQID NO:5, SEQID NO: 6, or SEQID NO:8. gtcatt.cgcggagggitttacgacgtgacgcgtgggtgcc.gcago atcct 45 In another embodiment, the isolated polynucleotide com prises a DNA sequence that is at least 95% homologous to the ggcggaalacctgat Ctttgttgaaagctggcc.gcgactgtacccagctgtt nucleic acid sequence of SEQID NO:4, SEQID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In another embodiment, the cgattic ctaccacc cct taagtgcc agggctgtgctagacaagttctaca isolated polynucleotide comprises a DNA sequence that is at tcggtgaagt catgtaaggcctggggacgagcagttcCttgttggctitt C 50 least 98% homologous to the nucleic acid sequence of SEQ ID NO:4, SEQID NO:5, SEQID NO: 6, or SEQID NO:8. gaagaggacacagaggagggit cagttctacacggtc.ct Caagaag.cgtgt In another embodiment, the isolated polynucleotide com ggagaagtactt Cagggagaacaagct caa.ccc.gcggcaa.caggcgc.cat prises a DNA sequence that is at least 60% identical to the nucleic acid sequence of SEQID NO:4, SEQID NO: 5, SEQ gtacgc.caagtc.gctgacCatCctggcgggcctggcgttgagcttctatg 55 ID NO: 6, or SEQ ID NO: 8. In another embodiment, the gtacgttctttgcc titcagcagcgcaccggccticgctgct cagcgctgtg isolated polynucleotide comprises a DNA sequence that is at least 70% identical to the nucleic acid sequence of SEQ ID Ctgctcggcatttgcatggcggaggtgggcgtgtc.cat catgcacgatgc NO: 4, SEQID NO: 5, SEQID NO: 6, or SEQID NO: 8. In another embodiment, the isolated polynucleotide comprises a Calaccacggcgcatttgc.ccgcaac acgtgggcct cqcatgcc ctgggcg 60 DNA sequence that is at least 75% identical to the nucleic acid ccacgctgga catcgtgggggcatcct cott catgtggcgc.ca.gcagcat sequence of SEQID NO: 4, SEQID NO:5, SEQID NO: 6, or SEQ ID NO: 8. In another embodiment, the isolated poly gtcgtgggccaccatgcatacaccalacgtggacggt caggaccCalgacct nucleotide comprises a DNA sequence that is at least 80% gcgagittaaggaccc.cgacgttcgc.cgcgtgacCaagttc.ca.gc.cccagc identical to the nucleic acid sequence of SEQID NO: 4, SEQ 65 ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In another agtcgtaccaggcgtaccagcacatctacctggcct tcctgtacggcctg embodiment, the isolated polynucleotide comprises a DNA sequence that is at least 85% identical to the nucleic acid US 9,315,837 B2 11 12 sequence of SEQID NO: 4, SEQID NO:5, SEQID NO: 6, or - Continued SEQ ID NO: 8. In another embodiment, the isolated poly LKGKVEQVSFLHWYHHASISTIWWAIAYWAPGGDAWYCCFLNSLVHVLMY nucleotide comprises a DNA sequence that is at least 90% identical to the nucleic acid sequence of SEQID NO: 4, SEQ TYYLLATLLGKDAKARRKYLWWGRYLTOFOMFOFWTMMLEAAYTWAYSPY ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In another PKFLSKLLFFYMITLLALFANFYAOKHGSSRAAKOKLQ embodiment, the isolated polynucleotide comprises a DNA In another embodiment, the elongase as described hereinid sequence that is at least 95% identical to the nucleic acid encoded by a polynucleotide comprising a DNA sequence sequence of SEQID NO: 4, SEQID NO:5, SEQID NO: 6, or comprising the sequence: SEQ ID NO: 8. In another embodiment, the isolated poly nucleotide comprises a DNA sequence that is at least 98% 10 identical to the nucleic acid sequence of SEQID NO: 4, SEQ (SEQ ID NO: 8) ID NO. 5, SEQ NO: 6, or SEQID NO: 8. atggcattgacggcggcctgg cacaagtacgacgct atcgittagt cqctt In another embodiment, the present invention comprises a desaturase or a nucleic acid molecule encoding the same tgttitt catggcttgcgcagggttggcctgcaa.gagattcaaggccacc combined with additional proteins and/or enzymes and/or 15 cct cqgtgat caccgc.ccacct tcc.ctt catago ct coccaacgc.cacaa substrates that are involved in the biosynthesis of VLC PUFA. In another embodiment, the present invention com gtgacgttcgtgctggcct atctgctgattgttgtctg.cggggttgcc.gc prises a composition comprising a desaturase or a nucleic totgcgtacgagaaagttctgtc.cgcacct Cogaggat.ccggcgtggctgc acid molecule encoding the same combined with additional proteins and/or enzymes and/or substrates that are involved in gactgcttgttgcaa.gc.gcaca acttggtgctaat cagccttagcgc.ctac the biosynthesis of VLC-PUFA. In another embodiment, the present invention comprises a transgenic plant comprising a atgtcc totgcc.gc.ctgctactatoctitggaaatacggctataggittittg desaturase or a nucleic acid molecule encoding the same gggcacaaactatagc.cccaaggagcggga catgggagggct Catctata combined with additional proteins and/or enzymes and/or substrates that are involved in the biosynthesis of VLC 25 CcttttacgtgtcCaagctgtacgagtttgttggatacgctgat catgctg PUFA. In another embodiment, the present invention com prises a transgenic alga comprising a desaturase or a nucleic ct Caagggcaaggtggagcaggttt Ctttitttgcacgt.ctaccaccacgc acid molecule encoding the same combined with additional tt CC at at CC acgatctggtgggcaatcgcatacgt.cgcacctggtggtg proteins and/or enzymes and/or substrates that are involved in the biosynthesis of VLC-PUFA. In another embodiment, the 30 acgc.ctgg tactgctgctt cotgaacticgctggit coacgtact catgitac present invention comprises a transfected or a transformed acatactacctgcttgcgacgctgctgggaaaggacgc.caaggcgcggcg cell comprising a desaturase or a nucleic acid molecule encoding the same combined with additional proteins and/or Caagtatttgttggtggggacgctacct cacticagttccagatgttcCagt enzymes and/or substrates that are involved in the biosynthe ttgttgacgatgatgctic gaggcagcgtacacttgggcct actict c cctac SiS of VLC-PUFA. 35 In another embodiment, the present invention comprises a cc caagtttittatcaaagctgctgttatttaCatgat cactctgttggcc desaturase or a nucleic acid molecule encoding the same combined with additional proteins and/or enzymes and/or Ctgtttgcaaact tctatgcacagaa.gcatggcagcago.cgggcagc.cala substrates that are involved in the biosynthesis of VLC gcaaaagctgcagtaa PUFA. In another embodiment, the present invention com 40 prises an algal desaturase or a nucleic acid molecule encoding In another embodiment, the present invention provides a the same combined with additional algal proteins and/or composition comprising a desaturase as described herein. In enzymes and/or substrates that are involved in the biosynthe another embodiment, the present invention provides a com sis of VLC-PUFA. In another embodiment, the present inven position comprising the desaturase as described herein and a tion provides that the alga is a microalga. In another embodi 45 VLC-PUFA elongase. In another embodiment, the present ment, the present invention comprises a microalgae invention provides a composition comprising a protein as desaturase or a nucleic acid molecule encoding the same described herein. In another embodiment, the present inven combined with additional microalgae proteins and/or tion provides a composition comprising the polynucleotide as enzymes and/or substrates that are involved in the biosynthe described herein. In another embodiment, the present inven SiS of VLC-PUFA. 50 tion provides a composition comprising a polynucleotide In another embodiment, the present invention provides that encoding an elongase and the polynucleotide as described algae proteins comprise an elongase. In another embodiment herein. In another embodiment, the present invention pro an elongase is described in PCT/IL2009/001117 which is vides a composition comprising a vector comprising the poly hereby incorporated in its entirety by reference. In another nucleotide as described herein. In another embodiment, the embodiment, the present invention provides that microalgae 55 present invention provides a composition comprising a vector proteins comprise the P incisa PiELO1 gene product. In comprising a polynucleotide encoding an elongase and a another embodiment, the present invention provides that polynucleotide as described herein. In another embodiment, elongase as described herein comprises the amino acid the present invention provides a composition comprising a Sequence: combination of Vectors which comprise polynucleotides 60 encoding an elongase and polynucleotides encoding desatu rases. In another embodiment, a composition Such as (SEO ID NO: 7) described herein comprises a carrier. In another embodiment, MALTAAWHKYDAIVSRFWFDGLRRVGLOEIOGHPSVITAHLPFIASPTPO a carrier stabilizes a protein or a nucleic acid molecule of the WTFWLAYLLIVWCGVAALRTRKSSAPREDPAWLRLLVOAHNLVLISLSAY invention. In another embodiment, one of skill in the art will 65 readily identify a known suitable carrier to be used with the MSSAACYYAWKYGYRFWGTNYSPKERDMGGLIYTFYWSKLYEFWD TLIML composition as described herein. In another embodiment, a carrier is a buffer such as but not limited to a phosphate buffer. US 9,315,837 B2 13 14 In another embodiment, one of skill in the art is able to plant source of ARA. In another embodiment, P incisa is the prepare a composition comprising a desaturase as described richest algal source of ARA. In another embodiment, algal herein. In another embodiment, one of skill in the artisable to derived genes such as described herein are more effective prepare a composition comprising a combination of elon alone or in combination than those of other sources. gases and desaturases as described herein. In another embodi In another embodiment, algae as described herein are ment, one of skill in the art is able to prepare a composition eukaryotic organisms. In another embodiment, algae as comprising a polynucleotide as described herein. In another described herein are photoautotrophic. In another embodi embodiment, one of skill in the art is able to prepare a com ment, algae as described herein are mixotrophic. In another position comprising a combination of polynucleotides, plas embodiment, algae as described herein are unicellular. In mids, vectors etc. as described herein. In another embodi 10 another embodiment, algae as described herein are multicel ment, the present invention provides a composition lular. In another embodiment, algae as described herein are comprising the protein as described herein to be used in Excavata algae. In another embodiment, algae as described foodstuffs, dietary Supplements or pharmaceutical composi herein are Rhizaria algae. In another embodiment, algae as tions. In another embodiment, the present invention provides described herein are Chromista algae. In another embodi a composition comprising the protein as described herein to 15 ment, algae as described herein are Alveolata algae. be used in industrial applications for the manufacturing of In another embodiment, an algal gene and protein of the VLC-PUFAs. In another embodiment, the present invention present invention is Superior when compared to its homo provides a composition comprising the VLC-PUFAs, the logues with respect to efficient production of PUFAs. In products of the enzymes of the present invention. In another another embodiment, transforming a first alga with an algal embodiment, a composition comprising VLC-PUFAs is used gene derived from a second alga Such as described herein in foodstuffs, dietary Supplements or pharmaceutical compo produces better results in comparison to fungal genes. In sitions. another embodiment, a second algal gene is a gene as In another embodiment, the invention includes a combina described herein. In another embodiment, a first and a second tion of A5, A6, and/or A12 desaturases. In another embodi algal are of different species. In another embodiment, trans ment, the invention includes a composition comprising the 25 forming a first alga with an algal gene derived from a second combination of A5, A6, and/or A12 desaturases. In another alga Such as described herein in combination with additional embodiment, the invention includes a composition compris genes that encode proteins that are involved in the biosynthe ing the combination of A5, A6, and/or A12 desaturases and sis of VLC-PUFA produces better results in comparison to either ()3 or ()6 C18 substrates. In another embodiment, the fungal or wild-type genes. In another embodiment, trans invention provides that a composition comprising the combi 30 forming an alga with an algal gene (Such as desaturase) nation of A5, A6, and/or A12 desaturases and either ()3 or (06 derived from a second alga Such as described herein in com C18 substrates yields DGLA, ARA, DHA and/or EPA. bination with an elongase as described herein produces better In another embodiment, the invention provides conjunc results in comparison to fungal or wild-type genes. In another tion of P incisa A12, A6, and A5 desaturases, which are set of embodiment, transforming a first alga with a combination of P incisa genes involved in the biosynthesis of ARA. In 35 algal genes derived from a second alga, a third alga, etc.. Such another embodiment, the invention provides conjunction of P as described herein produces better results (such as ARA incisa A12, A6, and A5 desaturase and A6 specific PUFA production) in comparison to fungal or wild-type genes. In elongase (as described herein), which are set of P incisa another embodiment, transforming a first alga with a combi genes involved in the biosynthesis of DGLA, ARA, DHA. nation of algal desaturase genes derived from a second alga EPA, or any combination thereof. 40 such as described herein produces better results (such as ARA In another embodiment, a desaturase as described herein production) in comparison to fungal or wild-type genes. In comprises three histidine rich motifs (his-boxes). In another another embodiment, P incisa is the second alga. In another embodiment, A6 (PiDesG) and A5 (PiDes5) desaturases com embodiment, P incisa is the source of choice for genes that prise fused cytochrome b5 at their N-terminus, Supporting are involved in the biosynthesis of VLC-PUFA. In another their microsomal localization. In another embodiment, A6 45 embodiment, P incisa is the source of choice for genes that (PiDesG) and A5 (PiDes5) desaturases comprise a HPGG are involved in the biosynthesis of ARA, DHA, EPA, DGLA, quartet along with four amino acids conserved in all cyto or any combination thereof. In another embodiment, P. chrome b5 fusion desaturases (FIG. 1). incisa-derived genes such as described herein are more effec In another embodiment, transforming a plant with an algal tive alone or in combination than those of other sources. derived gene such as described herein produces better results 50 In another embodiment, a DNA sequence as described in comparison to fungal genes. In another embodiment, trans herein such as but not limited to SEQID NO: 4-6 is used to forming a plant with an algal-derived gene Such as described engineer a transgenic organism. In another embodiment, herein in combination with additional genes that encode pro DNA sequences as described herein such as but not limited to teins that are involved in the biosynthesis of VLC-PUFA SEQID NO: 4-6 are used to engineer a transgenic organism produces better results in comparison to fungal or wild-type 55 or transform a cell. In another embodiment, DNA sequences genes. In another embodiment, transforming a plant with an as described herein such as but not limited to SEQID NO: 4-6 algal-derived gene Such as described herein (desaturase) in and 8 are used to engineer a transgenic organism or transform combination with an elongase as described herein produces a cell. In another embodiment, the DNA sequences comprise better results in comparison to fungal or wild-type genes. In the sequences provided in SEQID NO: 4-6 and 8 or variants another embodiment, transforming a plant with a combina 60 of these sequences due, for example: base Substitutions, dele tion of algal-derived genes such as described herein produces tions, and/or additions. better results (such as ARA production) in comparison to In another embodiment, the present invention provides fungal or wild-type genes. In another embodiment, trans transgenic plant oils enriched with VLC-PUFA. In another forming a plant with a combination of algal-derived desatu embodiment, the present invention provides transgenic alga rase genes such as described herein produces better results 65 oils enriched with VLC-PUFA. In another embodiment, the (such as ARA production) in comparison to fungal or wild present invention provides the reconstitution of C20-VLC type genes. In another embodiment, P incisa is the richest PUFA biosynthesis in oil-synthesizing seeds of higher plants. US 9,315,837 B2 15 16 In another embodiment, the present invention provides the protein/s as described herein in a cell, an alga, or a plant expanded use by enhancement of the levels of ARA, DGLA, under nitrogen starvation conditions. DHA, EPA, or a combination thereof in the transgenic plants. In another embodiment, a cell is a eukaryotic cell. In In another embodiment, the present invention provides an another embodiment, a cell is a prokaryotic cell. In another expression vector comprising the polynucleotideas described 5 embodiment, a cell is a plant cell. In another embodiment, a herein. In another embodiment, the present invention pro cell is an algal cell. In another embodiment, a cell is a trans vides a combination of expression vectors each comprising a fected cell. In another embodiment, a cell is transiently trans polynucleotide as described herein. In another embodiment, fected with a polynucleotide or a combination of polynucle the present invention provides an expression vector compris otides as described herein. In another embodiment, a cell is 10 stably transfected with a polynucleotide or a combination of ing a combination of polynucleotides as described herein. In polynucleotides as described herein. In another embodiment, another embodiment, the present invention provides a plant the present invention provides a method of enhancing oil specific expression vector comprising the polynucleotide or storage, EPA accumulation, DHA accumulation, ARA accu combination of polynucleotides as described herein. In mulation, DGLA accumulation, or a combination thereof in a another embodiment, the present invention provides an algal 15 cell, comprising the step of transforming a cell with a poly specific expression vector comprising the polynucleotide or nucleotide as described herein. In another embodiment, the combination of polynucleotides as described herein. In present invention provides a method of enhancing oil storage, another embodiment, the present invention provides a cell EPA accumulation, DHA accumulation, ARA accumulation, comprising the expression vector/s as described herein. In DGLA accumulation, or a combination thereof in a cell, another embodiment, the expression vector/s is contained comprising the step of transfecting a cell with a polynucle within an agrobacterium. In another embodiment, a cell is a otide as described herein. In another embodiment, the present plant cell oran algal cell. In another embodiment, a plant is an invention provides a method of enhancing oil storage, EPA oil crop. In another embodiment, the transformed plant is an accumulation, DHA accumulation, DGLA accumulation, oil crop. ARA accumulation, or a combination thereof in a cell, com In another embodiment, the present invention provides a 25 prising the step of transforming a cell with a combination of transgenic plant, a transgenic Seed, or a transgenic alga trans polynucleotides as described herein. In another embodiment, formed by a polynucleotide as described herein. In another the present invention provides a method of enhancing oil embodiment, the present invention provides a transgenic storage, EPA accumulation, DHA accumulation, ARA accu plant, a transgenic seed, or a transgenic alga transformed by mulation, DGLA accumulation, or a combination thereof in a any combination of polynucleotides as described herein. In 30 cellor a multicellular organism, comprising the step of trans another embodiment, the present invention provides that the forming a cell or a multicellular organism with a polynucle transgenic plant is a true-breeding for the polynucleotide?s as otide as described herein. In another embodiment, the present described herein. In another embodiment, the present inven invention provides a method of enhancing oil storage, EPA tion provides a transgenic seed, produced by a transgenic accumulation, DGLA accumulation, DHA accumulation, plant transformed by the polynucleotide/s as described 35 ARA accumulation, or a combination thereof in a multicel herein. In another embodiment, a transgenic plant, a trans lular organism, comprising the step of transforming a multi genic seed, or a transgenic alga as described herein produces cellular organism with a combination of polynucleotides as very long-chain polyunsaturated fatty acid (VLC-PUFA). In described herein. In another embodiment, the multicellular another embodiment, a transgenic plant, a transgenic seed, or organism or cell is grown under nitrogen starvation condi a transgenic alga as described herein produces arachidonic 40 tions, oleogenic conditions, or a combination thereof. acid. In another embodiment, a transgenic plant or a trans In another embodiment, transformation as used herein genic seed as described herein produces DHA. In another comprises “transduction'. In another embodiment, transfor embodiment, a transgenic plant, a transgenic seed, or a trans mation as used herein comprises transfection. In another genic alga as described herein produces DGLA. embodiment, transformation as used herein comprises "con In another embodiment, the present invention provides a 45 jugation'. In another embodiment, transformation as used method of producing very long-chain polyunsaturated fatty herein applies to eukaryotic and prokaryotic cells. In another acid (VLC-PUFA) in a plant, a plant cell, or an alga compris embodiment, transformation as used herein comprises the ing the step of transforming a plant, a plant cell, or an alga insertion of new genetic material into nonbacterial cells with a polynucleotideas described herein. In another embodi including animal and plant cells. ment, the present invention unexpectedly provides an utmost 50 In another embodiment, the present invention provides a efficient method of producing very long-chain polyunsatu method of enhancing oil storage, EPA accumulation, DHA rated fatty acid (VLC-PUFA) in a plant, a plant cell, or an alga accumulation, DGLA accumulation, ARA accumulation, or a comprising the step of transforming a plant, a plant cell, oran combination thereof in a plant cell, comprising the step of alga with a polynucleotide as described herein. In another transforming a plant cell with a polynucleotide as described embodiment, a VLC-PUFA is produced from gamma.-lino 55 herein. In another embodiment, the present invention pro lenic acid (GLA). vides a method of enhancing oil storage, EPA accumulation, In another embodiment, a VLC-PUFA is produced from DHA accumulation, ARA accumulation, DGLA accumula stearidonic acid (SDA). In another embodiment, a VLC tion, or a combination thereof in a plant cell, comprising the PUFA is produced from GLA, SDA, or their combination. In step of transforming a plant cell with a combination of poly another embodiment, a VLC-PUFA comprises 20 carbons. In 60 nucleotides as described herein. In another embodiment, the another embodiment, a VLC-PUFA is 20:306 or 20:4c03. In present invention provides a method of enhancing oil storage, another embodiment, a VLC-PUFA is produced by the pro EPA accumulation, DHA accumulation, ARA accumulation, tein/s as described herein in a cell or a plant, a plant cell, oran DGLA accumulation, or a combination thereof in a plant, alga. In another embodiment, a VLC-PUFA is produced by comprising the step of transforming a plant with a polynucle the protein/s as described herein in a cell, a plant, a plant cell, 65 otide as described herein. In another embodiment, the present or an alga under oleogenic conditions. In another embodi invention provides a method of enhancing oil storage, EPA ment, an unexpected amount of VLC-PUFA is produced by accumulation, DHA accumulation, ARA accumulation, US 9,315,837 B2 17 18 DGLA accumulation, or a combination thereof in a plant, an alga, comprising the step of transforming a cell, a plant, a comprising the step of transforming a plant with a combina plant cell, or an alga with a combination of exogenous poly tion of polynucleotides as described herein. In another nucleotides as described herein, thereby producing a VLC embodiment, the plant or plant cell is grown under nitrogen PUFA in a cell, a plant, a plant cell, or an alga. starvation conditions, oleogenic conditions, or a combination In another embodiment, the invention provides that a plant, thereof. a cell, a plant cell, or an alga as described herein is treated or In another embodiment, the invention further provides an supplemented with linoleic acid (LA; 18:2(O6), C.-linolenic engineered organism, such as a transgenic plant. In another acid (ALA; 18:303), oleic acid (18:1), dihomo-gamma-lino embodiment, the invention further provides an engineered lenic acid (20:3(O6), phosphatidylcholine (PC), diacylglyc organism, Such as a transgenic seed. In another embodiment, 10 eroltrimethylhomoserine (DGTS), phosphatidylethanola the invention further provides an engineered organism, Such mine (PE), or any combination thereof, before as a transgenic alga. In another embodiment, the invention transformation, after transformation, during transformation further provides an engineered organism, such as a transgenic or a combination thereof. animal. In another embodiment, an engineered organism is In another embodiment, the invention provides that the engineered to express a protein as described herein. In 15 VLC-PUFA is eicosapentaenoic acid (EPA, 20:5c)3). In another embodiment, an engineered organism is engineered another embodiment, the invention provides that the VLC to express a combination of proteins as described herein. In PUFA is docosahexaenoic acid (DHA, 22:6c)3). In another another embodiment, an engineered organism is engineered embodiment, the invention provides that the VLC-PUFA is to express elevated levels of the protein or a combination of arachidonic acid (ARA, 20:4()6). In another embodiment, the proteins. In another embodiment, an engineered plant as invention provides that a cell, a plant, or an alga transformed described herein is used for manufacturing desired PUFAs by a polynucleotide or a combination of polynucleotides as such as but not limited to ARA. In another embodiment, an described herein, is grown under oleogenic conditions. In engineered plant as described herein is used for manufactur another embodiment, the invention provides that a cell, a ing desired PUFAs such as ARA at a reduced cost. plant, or an alga transformed by a polynucleotide or a com In another embodiment, an engineered organism com 25 bination of polynucleotides as described herein, is grown prises a synthetic pathway for the production of a protein. In under nitrogen starvation conditions. another embodiment, an engineered organism comprising a In another embodiment, the invention provides that pro synthetic pathway for the production of the protein allows ducing very long-chain polyunsaturated fatty acid (VLC greater control over the production of PUFAs by the pathway PUFA) is enhancing oil storage, arachidonic acid accumula by an organism. In another embodiment, the pathway 30 tion, eicosapentaenoic acid accumulation, docosahexaenoic includes but is not limited to N-fatty acid desaturase, and/or acid accumulation, or a combination thereof that a cell, a N-fatty acid desaturase. plant, or an alga transformed by a polynucleotide or a com In another embodiment, an engineered cell, plant or seed bination of polynucleotides as described herein comprises an oligonucleotide as described herein. In another In another embodiment, a PUFA is di-homo-gamma-lino embodiment, an engineered plant or seed produces a protein 35 lenic acid, arachidonic acid, eicosapentaenoic acid, docosa as described herein and comprises an oligonucleotide as trienoic acid, docosatetraenoic acid, docosapentaenoic acid described herein. In another embodiment, an engineered or docosahexaenoic acid. In another embodiment, a PUFA is plant or seed produces proteins as described herein and com a 24 carbon fatty acid with at least 4 double bonds. prises oligonucleotides as described herein. In another embodiment, expression of the protein/s of the In another embodiment, the invention provides a method of 40 invention in plants or seed requires Subcloning an ORF/S producing very long-chain polyunsaturated fatty acid (VLC sequence encoding the protein/s into a plant expression vec PUFA) in a cell, a plant, a plant cell, oran alga, comprising the tor, which may comprise a viral 35S promoter, and a Nos step of transforming a plant, a plant cell, or an alga with a terminator. In another embodiment, a cassette or promoter/ polynucleotide as described herein, thereby producing a coding sequence?terminator is then be subcloned into the VLC-PUFA in a plant, a plant cell, or an alga. In another 45 plant binary transformation vector, and the resulting plasmid embodiment, the invention provides a method of producing introduced into Agrobacterium. In another embodiment, the very long-chain polyunsaturated fatty acid (VLC-PUFA) in a Agrobacterium strain transforms the plant. In another cell, a plant, a plant cell, or an alga, comprising the step of embodiment, the. Agrobacterium strain transforms the plant transforming a plant, a plant cell, or an alga with an exog by the vacuum-infiltration of inflorescences, and the seeds enous polynucleotide as described herein, thereby producing 50 harvested and plated onto selective media containing an anti a VLC-PUFA in a cell, a plant, a plant cell, or an alga. In biotic. In another embodiment, the plasmid confers resistance another embodiment, the invention provides a method of to an antibiotic, thus only transformed plant material will producing very long-chain polyunsaturated fatty acid (VLC grow in the presence of an antibiotic. In another embodiment, PUFA) in a cell, a plant, a plant cell, oran alga, comprising the resistant lines are identified and self-fertilized to produce step of transforming a plant, a plant cell, or an alga with a 55 homozygous material. In another embodiment, leaf material vector comprising an exogenous polynucleotide as described is analyzed for expression of the protein comprising desatu herein, thereby producing a VLC-PUFA in a cell; a plant, a rase activity. In another embodiment, leaf material is ana plant cell, or an alga. In another embodiment, the invention lyzed for expression of a combination of protein comprising provides a method of producing very long-chain polyunsatu desaturase and elongase activities. rated fatty acid (VLC-PUFA) in a cell, a plant, a plant cell, or 60 In some embodiments, the terms “protein', 'desaturase'. an alga, comprising the step of transforming a plant, a plant or “polypeptide' are used interchangeably. In some embodi cell, or an alga with a combination of vectors comprising a ments, “protein”, “desaturase', or “polypeptide' as used combination of exogenous polynucleotides as described herein encompasses native polypeptides (either degradation herein, thereby producing a VLC-PUFA in a cell, a plant, a products, synthetically synthesized polypeptides or recombi plant cell, or an alga. In another embodiment, the invention 65 nant polypeptides) and peptidomimetics (typically, syntheti provides a method of producing very long-chain polyunsatu cally synthesized polypeptides), as well as peptoids and semi rated fatty acid (VLC-PUFA) in a cell, a plant, a plant cell, or peptoids which are polypeptide analogs, which have, in some US 9,315,837 B2 19 20 embodiments, modifications rendering the polypeptides/pro In some embodiments, the polypeptides or proteins of teins even more stable while in a body or more capable of present invention are biochemically synthesized such as by penetrating into cells. using standard Solid phase techniques. In some embodiments, In some embodiments, modifications include, but are not these biochemical methods include exclusive solid phase Syn limited to N terminus modification, C terminus modification, thesis, partial Solid phase synthesis, fragment condensation, polypeptide bond modification, including, but not limited to, or classical solution synthesis. In some embodiments, these CH2-NH, CH2-S, CH2-S-O, O–C NH, CH2-O, CH2 methods are used when the polypeptide is relatively short CH2, S-C NH, CH=CH or CF=CH, backbone modifi (about 5-15 kDa) and/or when it cannot be produced by cations, and residue modification. Methods for preparing recombinant techniques (i.e., not encoded by a nucleic acid peptidomimetic compounds are well known in the art and are 10 specified, for example, in Quantitative Drug Design, C. A. sequence) and therefore involves different chemistry. Ramsden Gd. Chapter 17.2, F. Choplin Pergamon Press In some embodiments, Solid phase polypeptide or protein (1992), which is incorporated by reference as if fully set forth synthesis procedures are well known to one skilled in the art herein. Further details in this respect are provided here and further described by John Morrow Stewart and Janis inunder. 15 Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., In some embodiments, polypeptide bonds ( CO. NH ) Pierce Chemical Company, 1984). In some embodiments, within the polypeptide are substituted. In some embodiments, synthetic polypeptides or proteins are purified by preparative the polypeptide bonds are substituted by N-methylated bonds high performance liquid chromatography Creighton T. ( N(CH)—CO ). In some embodiments, the polypeptide (1983) Proteins, structures and molecular principles. WH bonds are substituted by ester bonds ( C(R)H C O— Freeman and Co. N.Y., and the composition of which can be O—C(R) N ). In some embodiments, the polypeptide confirmed via amino acid sequencing by methods known to bonds are substituted by ketomethylene bonds (—CO— one skilled in the art. CH, ). In some embodiments, the polypeptide bonds are In some embodiments, recombinant protein techniques are substituted by C.-azabonds ( NH N(R)—CO—), wherein used to generate the polypeptides of the present invention. In R is any alkyl, e.g., methyl, carbo bonds (—CH2—NH- ). In 25 Some embodiments, recombinant protein techniques are used some embodiments, the polypeptide bonds are substituted by for generation of relatively long polypeptides (e.g., longer hydroxyethylene bonds ( CH(OH)—CH2—). In some than 18-25 amino acid). In some embodiments, recombinant embodiments, the polypeptide bonds are substituted by thioa protein techniques are used for the generation of large mide bonds ( CS NH ). In some embodiments, the amounts of the polypeptide of the present invention. In some polypeptide bonds are substituted by olefinic double bonds 30 embodiments, recombinant techniques are described by Bit (—CH=CH-). In some embodiments, the polypeptide ter et al., (1987) Methods in Enzymol. 153:516-544, Studier bonds are substituted by retro amide bonds ( NH CO ). et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. In some embodiments, the polypeptide bonds are substituted (1984) Nature 310:511-5.14, Takamatsu et al. (1987) EMBO by polypeptide derivatives ( N(R)—CH2—CO ), wherein J. 6:307-311, Coruzzietal. (1984) EMBO.J. 3:1671-1680 and R is the “normal side chain, naturally presented on the car 35 Broglietal, (1984) Science 224:838-843, Gurley et al. (1986) bonatom. In some embodiments, these modifications occurat Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, any of the bonds along the polypeptide chain and even at 1988, Methods for Plant Molecular Biology, Academic Press, several (2-3 bonds) at the same time. NY. Section VIII, pp. 421-463. In Some embodiments, natural aromatic amino acids of the In one embodiment, a polypeptide or protein of the present polypeptide such as Trp, Tyr and Phe, be substituted for 40 invention is synthesized using a polynucleotide encoding a synthetic non-natural acid such as Phenylglycine, TIC, naph polypeptide or protein of the present invention. In some thylelanine (Nol), ring-methylated derivatives of Phe, halo embodiments, the polynucleotide encoding a polypeptide of genated derivatives of Phe oro-methyl-Tyr. In some embodi the present invention is ligated into an expression vector, ments, the polypeptides of the present invention include one comprising a transcriptional control of a cis-regulatory or more modified amino acid or one or more non-amino acid 45 sequence (e.g., promoter sequence). In some embodiments, monomers (e.g., fatty acid, complex carbohydrates, etc.). the cis-regulatory sequence is Suitable for directing constitu In one embodiment, “amino acid' or "amino acids' is tive expression of the polypeptide of the present invention. In understood to include the 20 naturally occurring amino acid; Some embodiments, the cis-regulatory sequence is Suitable those amino acid often modified post-translationally in Vivo, for directing tissue specific expression of the polypeptide of including, for example, hydroxyproline, phosphoserine and 50 the present invention. In some embodiments, the cis-regula phosphothreonine; and other unusual amino acid including, tory sequence is suitable for directing inducible expression of but not limited to,2-aminoadipic acid, hydroxylysine, isodes the polypeptide of the present invention. In another embodi mosine, nor-valine, nor-leucine and ornithine. In one embodi ment, a polypeptide is a protein comprising a desaturase as ment, "amino acid includes both D- and L-amino acid. described herein. In some embodiments, the polypeptides or proteins of the 55 In another embodiment, the polynucleotide comprises a present invention are utilized in a soluble form. In some genomic polynucleotide sequence. In another embodiment, embodiments, the polypeptides or proteins of the present the polynucleotide comprises a composite polynucleotide invention include one or more non-natural or natural polar Sequence. amino acid, including but not limited to serine and threonine In one embodiment, the phrase “a polynucleotide' refers to which are capable of increasing polypeptide or protein solu 60 a single or double stranded nucleic acid sequence which be bility due to their hydroxyl-containing side chain. isolated and provided in the form of an RNA sequence, a In some embodiments, the polypeptides or proteins of the complementary polynucleotide sequence (cDNA), a genomic present invention are utilized in a linear form, although it will polynucleotide sequence and/or a composite polynucleotide be appreciated by one skilled in the art that in cases where sequences (e.g., a combination of the above). cyclization does not severely interfere with polypeptide char 65 In one embodiment, “genomic polynucleotide sequence' acteristics, cyclic forms of the polypeptide can also be uti refers to a sequence derived (isolated) from a chromosome lized. and thus it represents a contiguous portion of a chromosome. US 9,315,837 B2 21 22 In one embodiment, "composite polynucleotide sequence' another embodiment, transient expression is from introduced refers to a sequence, which is at least partially complementary constructs which contain expression signals functional in the and at least partially genomic. In one embodiment, a compos host cell, but which constructs do not replicate and rarely ite sequence can include Some exonal sequences required to integrate in the host cell, or where the host cell is not prolif encode the polypeptide of the present invention, as well as erating. In another embodiment, transient expression also can Some intronic sequences interposing there between. In one be accomplished by inducing the activity of a regulatable embodiment, the intronic sequences can be of any source, promoter operably linked to the gene of interest. including of other genes, and typically will include conserved In another embodiment, stable expression is achieved by splicing signal sequences. In one embodiment, intronic introduction of a construct that integrates into the host sequences include cis acting expression regulatory elements. 10 In one embodiment, the polynucleotides of the present genome. In another embodiment, stable expression com invention further comprise a signal sequence encoding a sig prises autonomously replication within the host cell. In nal peptide for the secretion of the polypeptides of the present another embodiment, stable expression of the polynucleotide invention. In one embodiment, following expression, the sig of the invention is selected for through the use of a selectable nal peptides are cleaved from the precursor proteins resulting 15 marker located on or transfected with the expression con in the mature proteins. struct, followed by selection for cells expressing the marker. In some embodiments, polynucleotides of the present In another embodiment, stable expression results from inte invention are prepared using PCR techniques or any other gration, the site of the constructs integration can occur ran method or procedure known to one skilled in the art. In some domly within the host genome or can be targeted through the embodiments, the procedure involves the legation of two use constructs containing regions of homology with the host different DNA sequences (See, for example, “Current Proto genome Sufficient to target recombination with the host locus. cols in Molecular Biology', eds. Ausubel et al., John Wiley & In another embodiment, constructs are targeted to an endog Sons, 1992). enous locus, all or some of the transcriptional and transla In one embodiment, polynucleotides of the present inven tional regulatory regions can be provided by the endogenous tion are inserted into expression vectors (i.e., a nucleic acid 25 locus. construct) to enable expression of the recombinant polypep In another embodiment, an expression of a protein as tide. In one embodiment, the expression vector of the present described herein comprising desaturase activity includes invention includes additional sequences which render this functional transcriptional and translational initiation and ter vector Suitable for replication and integration in prokaryotes. mination regions that are operably, linked to the DNA encod In one embodiment, the expression vector of the present 30 ing the protein comprising a desaturase activity. In another invention includes additional sequences which render this embodiment, an expression of proteins as described herein vector suitable for replication and integration in eukaryotes. comprising various desaturase activities includes functional In one embodiment, the expression vector of the present transcriptional and translational initiation and termination invention includes a shuttle vector which renders this vector regions that are operably linked to the DNA encoding the Suitable for replication and integration in both prokaryotes 35 proteins comprising desaturase activity. In another embodi and eukaryotes. In some embodiments, cloning vectors com ment, an expression of proteins as described herein compris prise transcription and translation initiation sequences (e.g., ing desaturase and elongase activities includes functional promoters, enhancers) and transcription and translation ter transcriptional and translational initiation and termination minators (e.g., polyadenylation signals). regions that are operably linked to the DNA encoding each In one embodiment, a variety of prokaryotic or eukaryotic 40 protein comprising a desaturase or elongase activity. In cells can be used as host-expression systems to express the another embodiment, transcriptional and translational initia polypeptides of the present invention. In some embodiments, tion and termination regions are derived from a variety of these include, but are not limited to, microorganisms, such as nonexclusive sources, including the DNA to be expressed, bacteria transformed with a recombinant bacteriophage genes known or Suspected to be capable of expression in the DNA, plasmid DNA or cosmid DNA expression vector con 45 desired system, expression vectors, chemical synthesis, or taining the polypeptide coding sequence; yeast transformed from an endogenous locus in a host cell. In another embodi with recombinant yeast expression vectors containing the ment, expression in a plant tissue and/or plant part presents polypeptide coding sequence; plant cell systems infected certain efficiencies, particularly where the tissue or partis one with recombinant virus expression vectors (e.g., cauliflower which is harvested early, such as seed, leaves, fruits, flowers, mosaic virus, CaMV; tobacco mosaic virus, TMV) or trans 50 roots, etc. In another embodiment, expression can be targeted formed with recombinant plasmid expression vectors, such as to that location in a plant by utilizing specific regulatory Tiplasmid, containing the polypeptide coding sequence. sequences that are known to one of skill in the art. In another In some embodiments, non-bacterial expression systems embodiment, the expressed protein is an enzyme which pro are used (e.g., plant expression systems) to express the duces a product which may be incorporated, either directly or polypeptide of the present invention. 55 upon further modifications, into a fluid fraction from the host In one embodiment, yeast expression systems are used. In plant. In another embodiment, expression of a protein of the one embodiment, algae expression systems are used. In one invention, or antisense thereof, alters the levels of specific embodiment, plant expression systems are used. In one PUFAs, or derivatives thereof, found in plant parts and/or embodiment, a number of vectors containing constitutive or plant tissues. The desaturase coding region, in Some embodi inducible promoters can be used in yeast as disclosed in U.S. 60 ments, may be expressed either by itself or with other genes Pat. No. 5,932,447 which is hereby incorporated in its entirety Such as but not limited to elongase, in order to produce cells, by reference. In another embodiment, vectors which promote tissues, algae, and/or plant parts containing higher propor integration of foreign DNA sequences into the yeast chromo tions of desired PUFAs or in which the PUFA composition Some are used. more closely resembles that of human breast milk. In another In another embodiment, expression in a host cell can be 65 embodiment, the termination region is derived from the 3' accomplished in a transient or a stable fashion. In another region of the gene from which the initiation region was embodiment, a host cell is a cell as described herein. In obtained or from a different gene. In another embodiment, the US 9,315,837 B2 23 24 termination region usually is selected as a matter of conve In another embodiment, a variety of methods can be uti nience rather than because of any particular property. lized for the regeneration of plants from plant tissue. In In another embodiment, a plant or plant tissue is utilized as another embodiment, the method of regeneration will depend a host or host cell, respectively, for expression of the protein on the starting plant tissue and the particular plant species to of the invention which may, in turn, be utilized in the produc be regenerated. In another embodiment, methods for trans tion of polyunsaturated fatty acids. In another embodiment, forming dicots, primarily by use of Agrobacterium tumefa desired PUFAS are expressed in seed. In another embodi ciens, and obtaining transgenic plants are known in the art ment, methods of isolating seed oils are known in the art. In McCabe et al., Biol. Technology 6:923 (1988), Christouet al., another embodiment, seed oil components are manipulated Plant Physiol. 87:671-674 (1988)); Cheng et al., Plant Cell through the expression of the protein of the invention in order 10 Rep. 15:653657 (1996), McKently et al., Plant Cell Rep. 14:699-703 (1995)); Grant et al., Plant Cell Rep. 15:254-258, to provide seed oils that can be added to nutritional compo (1995). sitions, pharmaceutical compositions, animal feeds and cos In another embodiment, transformation of monocotyle metics. In another embodiment, a vector which comprises a dons using electroporation, particle bombardment, and Agro DNA sequence encoding the protein as described herein is 15 bacterium are known. In another embodiment, transforma linked to a promoter, and is introduced into the plant tissue or tion and plant regeneration are well established in the art. In plant for a time and under conditions Sufficient for expression another embodiment, assays for gene expression based on the of the protein. transient expression of cloned nucleic acid constructs have In another embodiment, a vector as described herein com been developed by introducing the nucleic acid molecules prises additional genes that encode other enzymes, for into plant cells by polyethylene glycol treatment, electropo example, elongase, A4-desaturase, a different A5-desaturase, ration, or particle bombardment (Marcotte et al., Nature 335: a different A6-desaturase, A10-desaturase, a different A12 454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989); desaturase. A 15-desaturase. A 17-desaturase. A 19-desaturase, McCarty et al., Cell 66:895-905 (1991); Hattoriet al., Genes or any combination thereof. In another embodiment, the plant Dev. 6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 tissue or plant produces the relevant substrate upon which the 25 (1990)). enzymes act or a vector encoding enzymes which produce In another embodiment, transient expression systems are Such Substrates may be introduced into the plant tissue, plant used to functionally dissect the oligonucleotides constructs. cellor plant. In another embodiment, a Substrate is sprayed on In another embodiment, practitioners are familiar with the plant tissues expressing the appropriate enzymes. In another standard resource materials which describe specific condi embodiment, the invention is directed to a transgenic plant 30 tions and procedures for the construction, manipulation and comprising the above-described vector, wherein expression isolation of macromolecules (e.g., DNA molecules, plasmids, of the nucleotide sequence of the vector results in production etc.), generation of recombinant organisms and the screening of a polyunsaturated fatty acid in, for example, the seeds of and isolating of clones, (see for example: Sambrook et al., the transgenic plant. Molecular Cloning: A Laboratory Manual, Cold Spring Har In another embodiment, the regeneration, development, 35 bor Press (1989); Maliga et al., Methods in Plant Molecular and cultivation of plants from single plant protoplast trans Biology, Cold Spring Harbor Press (1995); Birren et al., formants or from various transformed explants is well known Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, in the art (for example: Weissbach and Weissbach, In: Meth N.Y. (1998); Birren et al., Genome Analysis: Analyzing ods for Plant Molecular Biology, (Eds.), DNA, 2, Cold Spring Harbor, N.Y. (1998); Plant Molecular Academic Press, Inc. San Diego, Calif., (1988)). In another 40 Biology: A Laboratory Manual, eds. Clark, Springer, N.Y. embodiment, regeneration and growth process comprises the (1997)). steps of selection of transformed cells, culturing those indi In one embodiment, the expression vector of the present vidualized cells through the usual stages of embryonic devel invention can further include additional polynucleotide opment through the rooted plantlet stage. In another embodi sequences that allow, for example, the translation of several ment, transgenic embryos and seeds are similarly 45 proteins from a single mRNA Such as an internal ribosome regenerated. In another embodiment, resulting transgenic entry site (IBES) and sequences for genomic integration of rooted shoots are thereafter planted in an appropriate plant the promoter-chimeric polypeptide. growth medium Such as soil. In another embodiment, regen In Some embodiments, expression vectors containing regu eration and growth process of algae are known to one of skill latory elements from eukaryotic viruses such as retroviruses in the art. In another embodiment, identification, selection, of 50 are used by the present invention. In some embodiments, transgenic algae are known to one of skill in the art. recombinant viral vectors are useful for in vivo expression of In another embodiment, development or regeneration of the polypeptides of the present invention since they offer plants containing an exogenous polynucleotide as described advantages such as lateral infection and targeting specificity. herein encodes a protein as described herein and is well In one embodiment, lateral infection is inherent in the life known in the art. In another embodiment, development or 55 cycle of, for example, retrovirus, and is the process by which regeneration of algae containing an exogenous polynucle a single infected cell produces many progeny virions that bud otide as described herein encodes a protein as described off and infect neighboring cells. In one embodiment, the herein and is well known in the art. In another embodiment, result is that a large area becomes rapidly infected, most of the regenerated plants are self-pollinated to provide homozy which was not initially infected by the original viral particles. gous transgenic plants. In another embodiment, pollen 60 In one embodiment, viral vectors are produced that are unable obtained from the regenerated plants is crossed to seed-grown to spread laterally. In one embodiment, this characteristic can plants of agronomically important lines. In another embodi be useful if the desired purpose is to introduce a specified ment, pollen from plants of these important lines is used to gene into only a localized number of targeted cells. pollinate regenerated plants. In another embodiment, a trans In one embodiment, various methods can be used to intro genic plant of the present invention containing a desired 65 duce the expression vector of the present invention into cells. polypeptide is cultivated using methods well known to one Such methods are generally described in Sambrook et al., skilled in the art. Molecular Cloning: A Laboratory Manual, Cold Springs Har US 9,315,837 B2 25 26 bor Laboratory, New York (1989, 1992), in Ausubel et al., In one embodiment, following a predetermined time in Current Protocols in Molecular Biology, John Wiley and culture, recovery of the recombinant polypeptide or protein is Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene effected. Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., In one embodiment, the phrase “recovering the recombi Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vec- 5 nant polypeptide or protein' used herein refers to collecting tors: A Survey of Molecular Cloning Vectors and Their Uses, the whole fermentation medium containing the polypeptide Butterworths, Boston Mass. (1988) and Gilboa et al. Bio or protein and need not imply additional steps of separation or techniques 4 (6): 504-512, 1986 and include, for example, purification. stable or transient transfection, lipofection, electroporation In one embodiment, polypeptides or proteins of the present 10 invention are purified using a variety of standard protein and infection with recombinant viral vectors. In addition, see purification techniques, such as, but not limited to, affinity U.S. Pat. Nos. 5.464,764 and 5.487,992 for positive-negative chromatography, ion exchange chromatography, filtration, selection methods. electrophoresis, hydrophobic interaction chromatography, In one embodiment, plant expression vectors are used. In gel filtration chromatography, reverse phase chromatogra one embodiment, the expression of a polypeptide coding 15 phy, concanavalin A chromatography, chromatofocusing and sequence is driven by a number of promoters. In some differential solubilization. embodiments, viral promoters such as the 35S RNA and 19S In one embodiment, to facilitate recovery, the expressed RNA promoters of CaMV Brisson et al., Nature 310:511 coding sequence can be engineered to encode the polypeptide 514 (1984), or the coat protein promoter to TMV Takamatsu or proteins of the present invention and fused cleavable moi et al., EMBO J. 6:307-311 (1987) are used. In another 20 ety. In one embodiment, a fusion protein can be designed so embodiment, plant promoters are used Such as, for example, that the polypeptide or protein can be readily isolated by the small subunit of RUBISCO Coruzzi et al., EMBO J. affinity chromatography; e.g., by immobilization on a column 3:1671-1680 (1984); and Brogliet al., Science 224:838-843 specific for the cleavable moiety. In one embodiment, a cleav (1984) or heat shock promoters, e.g., soybean hsp17.5-E or age site is engineered between the polypeptide or protein and hsp17.3-B Gurley et al., Mol. Cell. Biol. 6:559-565 (1986). 25 the cleavable moiety and the polypeptide or protein can be In one embodiment, constructs are introduced into plant cells released from the chromatographic column by treatment with using Tiplasmid, Riplasmid, plant viral vectors, direct DNA an appropriate enzyme or agent that specifically cleaves the transformation, microinjection, electroporation and other fusion protein at this site e.g., see Booth et al., Immunol. techniques well known to the skilled artisan. See, for Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990). example, Weissbach & Weissbach Methods for Plant 30 In one embodiment, the polypeptide or protein of the Molecular Biology, Academic Press, NY. Section VIII, pp present invention is retrieved in “substantially pure' form. 421-463(1988). Other expression systems such as insects In one embodiment, the phrase “substantially pure” refers and mammalian host cell systems, which are well known in to a purity that allows for the effective use of the protein in the the art, can also be used by the present invention. 35 applications described herein. It will be appreciated that other than containing the neces In one embodiment, the polypeptide or protein of the sary elements for the transcription and translation of the present invention can also be synthesized using in vitro inserted coding sequence (encoding the polypeptide or pro expression systems. In one embodiment, in vitro synthesis tein), the expression construct of the present invention can methods are well known in the art and the components of the also include sequences engineered to optimize stability, pro- 40 system are commercially available. duction, purification, yield or activity of the expressed In another embodiment, the invention comprises a process polypeptide or protein. for making a very long-chain polyunsaturated fatty acid pro In some embodiments, transformed cells are cultured duced by the protein or combination of proteins of the inven under effective conditions, which allow for the expression of tion in a cell as described herein. In another embodiment, the high amounts of recombinant polypeptide or protein. In some 45 resulting very long-chain polyunsaturated fatty acid pro embodiments, effective culture conditions include, but are duced by the transgenic cell or organism as described herein not limited to, effective media, bioreactor, temperature, pH is utilized as a food additive. In another embodiment, a very and oxygen conditions that permit protein production. In one long-chain polyunsaturated fatty acid produced by the trans embodiment, an effective medium refers to any medium in genic cell or organism as described herein is utilized as a which a cell is cultured to produce the recombinant polypep- 50 Supplement. In another embodiment, a very long-chain poly tide or protein of the present invention. In some embodi unsaturated fatty acid produced by the transgenic cell or ments, a medium typically includes an aqueous solution hav organism as described herein is administered to a human ing assimilable carbon, nitrogen and phosphate sources, and Subject. In another embodiment, a very long-chain polyun appropriate salts, minerals, metals and other nutrients, such as saturated fatty acid produced by the transgenic cell or organ Vitamins. In some embodiments, cells of the present invention 55 ism as described herein is administered to a baby. In another can be cultured in conventional fermentation bioreactors, embodiment, a very long-chain polyunsaturated fatty acid shake flasks, test tubes, microtiter dishes and petri plates. In produced by the transgenic cell or organism as described Some embodiments, culturing is carried out at a temperature, herein is administered to an infant. In another embodiment, a pH and oxygen content appropriate for a recombinant cell. In very long-chain polyunsaturated fatty acid produced by the Some embodiments, culturing conditions are within the 60 transgenic cell or organism as described herein is adminis expertise of one of ordinary skill in the art. tered to an animal. In another embodiment, a very long-chain In some embodiments, depending on the vector and host polyunsaturated fatty acid produced by the transgenic cell or system used for production, resultant polypeptides or pro organism as described herein is administered to a mammal. In teins of the present invention either remain within the recom another embodiment, a very long-chain polyunsaturated fatty binant cell, secreted into the fermentation medium, secreted 65 acid produced by the transgenic cell or organism as described into a space between two cellular membranes, or retained on herein is administered to a farm animal, a rodent, a pet, or a the outer surface of a cell or viral membrane. lab animal. US 9,315,837 B2 27 28 In another embodiment, the described pharmaceutical and per, Zinc, Selenium, iodine, and Vitamins A, E, D, C, and the nutritional compositions are utilized in connection with ani B complex. Other such vitamins and minerals may also be mals (i.e., domestic or non-domestic), as well as humans, as added. animals experience many of the same needs and conditions as In another embodiment, components utilized in the nutri humans. For example, the oil oracids of the present invention tional compositions of the present invention will be of semi may be utilized in animal or aquaculture feed Supplements, purified or purified origin. By semi-purified or purified is animal feed Substitutes, animal vitamins or in animal topical meant a material which has been prepared by purification of ointments. a natural material or by Synthesis. In another embodiment, In another embodiment, a very long-chain polyunsaturated nutritional compositions of the present invention include but 10 are not limited to infant formulas, dietary Supplements, fatty acid produced by a protein or a combination of proteins dietary Substitutes, and rehydration compositions. In another of the invention is utilized in an infant formula. In another embodiment, a nutritional composition of the present inven embodiment, a very long-chain polyunsaturated fatty acid tion may also be added to food even when Supplementation of produced by a protein or a combination of proteins of the the diet is not required. In another embodiment, a composi invention is administered to a Subject having a deficiency in 15 tion is added to food of any type including but not limited to very long-chain polyunsaturated fatty acid. In another margarines, modified butters, cheeses, milk, yogurt, choco embodiment, a very long-chain polyunsaturated fatty acid is late, candy, Snacks, salad oils, cooking oils, cooking fats, a polyunsaturated C20 fatty acid. meats, fish and beverages. In another embodiment, the isolated protein comprising In another embodiment, a nutritional composition is an desaturase activity is used indirectly or directly in the produc enteral nutritional product. In another embodiment, a nutri tion of polyunsaturated fatty acids. In another embodiment, tional composition is an adult or pediatric enteral nutritional the isolated protein or a combination of isolated proteins product. In another embodiment, a composition is adminis comprising desaturase and/or desaturasefelongase activities tered to adults or children experiencing stress or having spe are used indirectly or directly in the production of polyun cialized needs due to chronic or acute disease states. In saturated fatty acids. In another embodiment, “Directly is 25 another embodiment, a composition comprises, in addition to meant to encompass the situation where the enzyme directly polyunsaturated fatty acids produced in accordance with the desaturates the acid. In another embodiment, the latter of present invention, macronutrients, vitamins and minerals as which is utilized in a composition. In another embodiment, described above. In another embodiment, the macronutrients “Indirectly' is meant to encompass the situation where an may be present in amounts equivalent to those present in acid is converted to another acid (i.e., a pathway intermediate) 30 human milk or on an energy basis, i.e., on a per calorie basis. by the enzyme and then the latter acid is converted to another In another embodiment, the present invention includes an acid by use of a non-desaturase enzyme. In another embodi enteral formula comprising polyunsaturated fatty acids pro ment, a very long-chain polyunsaturated fatty acid produced duced in accordance with the present invention. In another either directly or indirectly is added to a nutritional compo embodiment, an enteral formula is sterilized and Subse sition, pharmaceutical compositions, cosmetics, and animal 35 quently utilized on a ready-to-feed basis or stored in a con feeds, all of which are encompassed by the present invention. centrated liquid or powder. In another embodiment, a powder In another embodiment, nutritional compositions include is prepared by spray drying the formula prepared as indicated any food or preparation for human or animal consumption above, and reconstituting it by rehydrating the concentrate. In including for enteral or parenteral consumption, which when another embodiment, the present invention includes an adult taken into the body (a) serve to nourish or build up tissues or 40 and pediatric nutritional formulas. In another embodiment, Supply energy and/or (b) maintain, restore or Support adult and pediatric nutritional formulas are known in the art adequate nutritional status or metabolic functions. In another and are commercially available (e.g., Similac R. Ensure R, embodiment, the nutritional composition of the present Jevity(R) and AlimentumR) from Ross Products Division, invention comprises at least one oil or acid produced directly Abbott Laboratories). In another embodiment, an oil or acid or indirectly by use of the protein of the invention and may 45 produce in accordance with the present invention may be add either be in a solid or liquid form. In another embodiment, the to any of these formulas. composition includes edible macronutrients, vitamins and In another embodiment, a nutritional formula comprises minerals in amounts desired for a particular use. In another macronutrients, vitamins, and minerals, as provided herein, embodiment, the amount of Such ingredients will vary in addition to the PUFAs produced in accordance with the depending on whether the composition is intended for use 50 present invention. In another embodiment, the presence of with normal, healthy infants, children or adults having spe additional components helps the individual ingest the mini cialized needs such as those which accompany certain meta mum daily requirements of these elements. In another bolic conditions (e.g., metabolic disorders). embodiment, an adult and pediatric nutritional formulascom In another embodiment, the macronutrients include edible prises the PUFAs as described herein and zinc, copper, folic fats, carbohydrates and proteins. In another embodiment, 55 acid and antioxidants, or any combination thereof. In another edible fats include but are not limited to coconut oil, soy oil, embodiment, PUFAs produced in accordance with the and mono- and diglycerides. In another embodiment, carbo present invention, or derivatives thereof, are added to a hydrates include but are not limited to glucose, edible lactose dietary Substitute or Supplement, particularly an infant for and hydrolyzed search. In another embodiment, proteins mula, for patients undergoing intravenous feeding or for pre which are utilized in the nutritional composition of the inven 60 venting or treating malnutrition or other conditions or disease tion include but are not limited to soy proteins, electrodialy states. In another embodiment, PUFAs produced in accor sed whey, electrodialysed skim milk, milk whey, or the dance with the present invention are used to alter, the com hydrolysates of these proteins. position of infant formulas in order to better replicate the In another embodiment, vitamins and minerals are added to PUFA content of human breast milk or to alter the presence of the nutritional compositions of the present invention and 65 PUFAs normally found in a non-human mammal’s milk. include but are not limited to: calcium, phosphorus, potas In another embodiment, parenteral nutritional composi sium, Sodium, chloride, magnesium, manganese, iron, cop tions comprising from about 2 to about 30 weight percent US 9,315,837 B2 29 30 fatty acids calculated as triglycerides are encompassed by the In one embodiment, compositions for use in accordance present invention. In another embodiment, other vitamins, with the present invention is formulated in conventional man particularly fat-soluble vitamins such as vitamin A, D, E and ner using one or more physiologically acceptable carriers L-carnitine are also included. In another embodiment, a pre comprising excipients and auxiliaries, which facilitate pro servative Such as alpha-tocopherol is added in an amount of 5 cessing of the proteins/polynucleotides into preparations. In about 0.05-0.5% by weight. one embodiment, formulation is dependent upon the method In another embodiment, the present invention includes a of administration chosen. PUFA produced in accordance with the present invention or The compositions also comprise, in some embodiments, host cells containing them, used as animal food Supplements preservatives, such as benzalkonium chloride and thimerosal to alter an animal's tissue or milk fatty acid composition to 10 and the like; chelating agents, such as edetate Sodium and one more desirable for human or animal consumption. others; buffers such as phosphate, citrate and acetate; tonicity In one embodiment, the polypeptides or protein of the agents such as Sodium chloride, potassium chloride, glycerin, present invention can be provided to the individual perse. In mannitol and others; antioxidants such as ascorbic acid, ace one embodiment, the polypeptides or proteins of the present tylcystine, sodium metabisulfote and others; aromatic agents; invention can be provided to the individual as part of a phar 15 Viscosity adjustors, such as polymers, including cellulose and maceutical composition where it is mixed with a pharmaceu derivatives thereof; and polyvinyl alcohol and acid and bases tically acceptable carrier. to adjust the pH of these aqueous compositions as needed. In one embodiment, a “pharmaceutical composition' The compositions also comprise, in Some embodiments, local refers to a preparation of one or more of the active ingredients anesthetics or other actives. The compositions can be used as described herein with other chemical components such as sprays, mists, drops, and the like. physiologically Suitable carriers and excipients. The purpose Additionally, Suspensions of the active ingredients, in of a pharmaceutical composition is to facilitate administra Some embodiments, are prepared as appropriate oily or water tion of a compound to an organism. In one embodiment, based suspensions. Suitable lipophilic solvents or vehicles “active ingredient” refers to the polypeptide or protein include, in some embodiments, fatty oils such as sesame oil, sequence of interest. 25 or synthetic fatty acid esters such as ethyl oleate, triglycerides In one embodiment, the present invention provides com or liposomes. Aqueous injection Suspensions contain, in bined preparations. In one embodiment, “a combined prepa Some embodiments, Substances, which increase the viscosity ration' defines especially a “kit of parts' in the sense that the of the Suspension, Such as sodium carboxymethyl cellulose, combination partners as defined above can be dosed indepen Sorbitol or dextran. In another embodiment, the Suspension dently or by use of different fixed combinations with distin 30 also contains suitable stabilizers or agents, which increase the guished amounts of the combination partners i.e., simulta solubility of the active ingredients to allow for the preparation neously, concurrently, separately or sequentially. In some of highly concentrated solutions. embodiments, the parts of the kit of parts can then, e.g., be In another embodiment, the proteins as described herein administered simultaneously or chronologically staggered, can be delivered in a vesicle, in particular a liposome (see that is at different time points and with equal or different time 35 Langer, Science 249:1527-1533 (1990); Treat et al., in Lipo intervals for any part of the kit of parts. The ratio of the total Somes in the Therapy of Infectious Disease and Cancer, amounts of the combination partners, in some embodiments, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353–365 can be administered in the combined preparation. In one (1989); Lopez-Berestein, ibid., pp. 317-327; see generally embodiment, the combined preparation can be varied, e.g., in ibid). order to cope with the needs of a patient subpopulation to be 40 In some embodiments, the protein as described herein is in treated or the needs of the single patient which different needs powder form for constitution with a suitable vehicle, e.g., can be due to a particular disease, severity of a disease, age, sterile, pyrogen-free water based solution, before use. In sex, or body weight as can be readily made by a person skilled another embodiment, compositions are contained in a con in the art. tainer with attached atomizing means. In one embodiment, the phrase “physiologically accept 45 In some embodiments, compositions Suitable for use in able carrier refers to a carrier or a diluent that does not cause context of the present invention include compositions significant irritation to a tissue Such as a plant tissue or a cell wherein the proteins or oligonucleotides are contained in an Such as a plant cell; and does not abrogate the biological amount effective to achieve the intended purpose. In one activity and properties of the protein or polynucleotide of the embodiment, determination of the effective amount is well invention. An adjuvant is included under these phrases. In one 50 within the capability of those skilled in the art. embodiment, one of the ingredients included in the physi Some examples of Substances which can serve as carriers ologically acceptable carrier can be for example polyethylene or components thereof are Sugars, such as lactose, glucose glycol (PEG), a biocompatible polymer with a wide range of and Sucrose; starches, such as corn starch and potato starch; solubility in both organic and aqueous media (Mutter et al. cellulose and its derivatives, such as sodium carboxymethyl (1979). 55 cellulose, ethyl cellulose, and methyl cellulose; powdered In one embodiment, “excipient” refers to an inert substance tragacanth; malt, gelatin; talc.; Solid lubricants, such as Stearic added to the composition to further facilitate administration acid and magnesium Stearate; calcium sulfate; vegetable oils, of an active ingredient. In one embodiment, excipients Such as peanut oil, cottonseed oil, sesame oil, olive oil, corn include calcium carbonate, calcium phosphate, various Sug oil and oil of theobroma; polyols such as propylene glycol, ars and types of Starch, cellulose derivatives, gelatin, Veg 60 glycerine, Sorbitol, mannitol, and polyethylene glycol; alg etable oils and polyethylene glycols. inic acid; emulsifiers, such as the Tween brand emulsifiers; Techniques for formulation and administration of peptide wetting agents, such sodium lauryl Sulfate; coloring agents; to plants or in-vitro are known to one of skill in the art. flavoring agents; tableting agents, stabilizers; antioxidants; In one embodiment, compositions of the present invention preservatives; pyrogen-free water, isotonic saline; and phos are manufactured by processes well known in the art, e.g., by 65 phate buffer solutions. The choice of a pharmaceutically means of conventional mixing, dissolving, or lyophilizing acceptable carrier to be used in conjunction with the com processes. pound is basically determined by the way the compound is to US 9,315,837 B2 31 32 be administered. If the subject compound is to be injected, in as delineated hereinabove and as claimed in the claims sec one embodiment, the pharmaceutically-acceptable carrier is tion below finds experimental support in the following sterile, physiological saline, with a blood-compatible Sus examples. pending agent, the pH of which has been adjusted to about 74. EXAMPLES In addition, the compositions further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, Generally, the nomenclature used herein and the laboratory guar gum, hydroxypropyl cellulose, hydroxypropyl methyl procedures utilized in the present invention include molecu cellulose, povidone), disintegrating agents (e.g., cornstarch, lar, biochemical, microbiological and recombinant DNA potato starch, alginic acid, silicon dioxide, croscarmelose 10 techniques. Such techniques are thoroughly explained in the Sodium, crospovidone, guar gum, Sodium starch glycolate), literature. See, for example, “Molecular Cloning: A labora tory Manual” Sambrook et al., (1989): “Current Protocols in buffers (e.g., Tris-HCl, acetate, phosphate) of various pH and Molecular Biology Volumes I-III Ausubel, R. M., ed. ionic strength, additives such as albuminor gelatin to prevent (1994); Ausubel et al., “Current Protocols in Molecular Biol absorption to Surfaces, detergents (e.g., Tween 20, Tween 80, 15 ogy”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, Pluronic F68, bile acid salts), protease inhibitors, surfactants “A Practical Guide to Molecular Cloning”, John Wiley & (e.g., sodium lauryl Sulfate), permeation enhancers, solubi Sons, New York (1988); Watson et al., “Recombinant DNA, lizing agents (e.g., glycerol, polyethylene glycerol), anti-oxi Scientific American Books, New York; Birren et al. (eds) dants (e.g., ascorbic acid, sodium metabisulfite, butylated "Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, Cold Spring Harbor Laboratory Press, New York (1998); hyroxypropylmethyl cellulose), Viscosity increasing agents methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683, (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, 202; 4,801,531; 5,192,659 and 5.272,057; “Cell Biology: A guar gum), Sweeteners (e.g., aspartame, citric acid), preser Laboratory Handbook'', Volumes I-III Cellis, J. E., ed. Vatives (e.g., Thimerosal, benzyl alcohol, parabens), lubri (1994): “Culture of Animal Cells—A Manual of Basic Tech cants (e.g., Stearic acid, magnesium Stearate, polyethylene 25 nique” by Freshney, Wiley-Liss, N.Y. (1994), Third Edition: glycol, Sodium lauryl Sulfate), flow-aids (e.g., colloidal sili “Current Protocols in Immunology” Volumes I-III Coligan.J. con dioxide), plasticizers (e.g., diethyl phthalate, triethyl cit E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immu rate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, nology” (8th Edition), Appleton & Lange, Norwalk, Conn. Sodium lauryl Sulfate), polymer coatings (e.g., poloxamers or (1994); Mishell and Shiigi (eds), “Selected Methods in Cel poloxamines), coating and film forming agents (e.g., ethyl 30 lular Immunology”. W. H. Freeman and Co., New York cellulose, acrylates, polymethacrylates) and/or adjuvants. (1980); available immunoassays are extensively described in The compositions also include incorporation of the pro the patent and scientific literature, see, for example, U.S. Pat. teins or oligonucleotides of the invention into or onto particu Nos. 3,791,932; 3,839,1533,850,752; 3,850,578; 3,853.987; late preparations of polymeric compounds such as polylactic 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; acid, polglycolic acid, hydrogels, etc., or onto liposomes, 35 3.996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and microemulsions, micelles, unilamellar or multilamellar 5,281.521; “Oligonucleotide Synthesis' Gait, M. J., ed. vesicles, erythrocyte ghosts, or spheroplasts.) Such composi (1984): “Nucleic Acid Hybridization’ Hames, B. D., and tions will influence the physical state, solubility, stability, rate Higgins S. J., eds. (1985): “Transcription and Translation' of in vivo release, and rate of in vivo clearance. Hames, B. D., and Higgins S. J., eds. (1984); 'Animal Cell Also comprehended by the invention are particulate com 40 Culture' Freshney, R.I., ed. (1986): “Immobilized Cells and positions coated with polymers (e.g. poloxamers or poloxam Enzymes' IRL Press, (1986): “A Practical Guide to Molecu ines) and the proteins or oligonucleotides of the invention lar Cloning Perbal, B., (1984) and “Methods in Enzymol coupled to antibodies directed against tissue-specific recep ogy” Vol. 1-317, Academic Press: “PCR Protocols: A Guide tors, ligands or antigens or coupled to ligands of tissue-spe To Methods And Applications'. Academic Press, San Diego, cific receptors. 45 Calif. (1990); Marshak et al., “Strategies for Protein Purifi In some embodiments, the proteins or oligonucleotides of cation and Characterization—A Laboratory Course Manual the invention modified by the covalent attachment of water CSHL Press (1996); all of which are incorporated by refer soluble polymers such as polyethylene glycol, copolymers of ence. Other general references are provided throughout this polyethylene, glycol and polypropylene glycol, carboxym document. ethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrroli 50 done or polyproline. In another embodiment, the modified Experimental Procedures proteins or oligonucleotides of the invention exhibit substan tially longer half-lives in blood following intravenous injec Strains and Growth Conditions tion than do the corresponding unmodified compounds. In one embodiment, modifications also increase the proteins or 55 Axenic cultures of P incisa were cultivated on BG-11 oligonucleotides solubility in aqueous solution, eliminate nutrient medium in 250 ml Erlenmeyer glass flasks in an aggregation, enhance the physical and chemical stability of incubator shaker at controlled temperature (25.degree. C.) the compound, and greatly reduce the immunogenicity and and illumination (115 mulmol quantam S') under an air/ reactivity of the compound. In another embodiment, the CO atmosphere (99:1, V/v) and a speed of 170 rpm. For desired in vivo biological activity is achieved by the admin 60 N-starvation experiments, cells of daily-diluted cultures were istration of Such polymer-compound abducts less frequently collected by centrifugation, washed three times in sterile or in lower doses than with the unmodified compound. DDW and resuspended in N-free BG 11 medium. To prepare Additional objects, advantages, and novel features of the N-free BG-11 medium, sodium nitrate was omitted and ferric present invention will become apparent to one ordinarily ammonium citrate was substituted with ferric citrate. Biom skilled in the art upon examination of the following examples, 65 ass was sampled at time 0, and in 1.5, 3, 7 and 14 days from which are not intended to be limiting. Additionally, each of the onset of N-starvation for determination of growth param the various embodiments and aspects of the present invention eters, and was further used for fatty acid analysis and RNA US 9,315,837 B2 33 34 isolation. Duplicate samples were collected from 3 separate Thermo Scientific, Surrey, UK) using the degenerated prim flasks for each time point and measurement. ers listed in the Table 1. To generate the full-length cDNAs, 3'- Growth Parameters and 5'-rapid amplification of the cDNA ends (RACE) was Dry weight and chlorophyll contents were determined as performed using a BD SmartTM RACE cDNA Amplification previously described in A. E. Solovchenko, I. Khozin-Gold- 5 Kit (BD Biosciences Clontech, Foster City, Calif., USA). berg, Z. Cohen, M. N. Merzlyak, Carotenoid-to-chlorophyll Gene specific primers were designed (Table 1) and RACE ratio as a proxy for assay of total fatty acids and arachidonic PCR reactions were conducted using 5' and 3'-RACE-Ready acid content in the green micro-alga, Parietochloris incisa, J. cDNAs made from 1.mu.g. total RNA of N-starved cells with Appl. Phycol. (2008) 361-366. 50.times. BD Advantage 2 polymerase mix (Clontech Labo RNA Isolation 10 ratories Inc., Mountain View, Calif., USA). The PCR prod Aliquots of the cultures were filtered through a glass fiber ucts of the expected sizes were excised, purified from the gel filter (GF-52, Schleicher & Schuell, Germany); cells were (Nucleo Spin Extract II purification kit, Machery-Nagel, collected by Scraping and immediately flash-frozen in liquid Duren, Germany) and ligated into a pGEMT-Easy vector nitrogen and stored at -80.degree. C. for further use. Total (Promega, Madison, Wis., USA). The full-length cDNAs RNA was isolated by the procedure described by Bekesiova et 15 were assembled based on the sequences of the 5' and 3' RACE al. (I. Bekesiova, J. P. Nap, L. Mlynarova, Isolation of high fragments. TABLE 1.

Primers used for obtaining partial, 5' and 3' end fragments of actin, A12, A6 and A5 desaturase genes of E. incisa followed by full-length assembly Gene Forward/Reverse primer (Sequence 5' to 3') SEQ ID NO : Primers used for partial sequence DesA12 CAC MYC VTS THC VWG CTG CTG WWE CCC CAC (FWD) 9 CTG CCC GAA GTT GAC CGC GGC GTG CTG (REV) 1O DesA6 TGG TGG AAR CAY AAR CAY AAY (FWD) 11 GCG AGG GAT CCA AGG RAA NAR RTG RTG YTC (REV) 12 DesA5 ATH RAI GRI AAR GTI TAY GAY GT (FWD) 13 GGI AYI KWI TSD ATR TCI GGRTC (REV) 14 Actin AGA TCT GGC ACC ACA CCT TCT TCA (FWD) 15 TGT TOT TGT AGA. GGT CCT TCC GGA (REV) 16 Primers used for 5' and 3' RACE amplification

DesA12 CCACATAGCGGCACAGGCTGAAATC (FWD) 17 GCTCTGGGAGGATTTCAGCCTGTGC (REV) 18 DesA6 GACACAATCTGGGCCGTCACAAAGTC (FWD) 19 GGACTTTGTGACGGCCCAGATTGTGTC (REV) 2O DesA5 ACTGACCCTCCTCTGTGTCCTCTTCG (FWD) 21 TGTACGCCAAGTCGCTGACCATCC (REV) 22 Primers used for full-length cloning and yeast transformations

Restriction sites */SEQ ID NO :

DesA12 TGGAATTCAAAATGGGGAAAGGAGGCTG (FWD) EcoRIA23 CTGTCTAGATCAAGCGCGGAACCACAGG Xbal/24

DesA6 TCGAATTCAAAATGTGCCAGGGACAGG (FWD) EcoRI/25 GGCTCTAGACTAGGCCTCAGCTGCCACG Xbal/26

DesA5 CCAAAGCTTAAAATGATGGCTGTAACAGA (FWD) HindIII/27 GCTCTAGACTATCCCACGGTGGCCA Xbal/28

55 quality DNA and RNA from leaves of the carnivorous plant Expression and Functional Characterization in the Yeast Sac Drosera rotundifolia, Plant. Mol. Biol. Rep. 17 (1999) 269 charomyces Cerevisiae 277), with minor modifications. Three independent RNA iso The open reading frames (ORFs) encoding for the A2, A6, lations were conducted for each time point. The total RNA 60 and A5 desaturases were amplified using Pful Jltra II fusion samples were treated with RNAase-free Baseline-ZEROTM HSDNA polymerase (Stratagene, LaJolla, Calif., USA) with DNAase (Epicentre Technologies, Madison, Wis., USA) the respective primer pairs (Table 1). The forward primers before being used in cDNA synthesis for real-time PCR contained a restriction site (underlined) and a yeast transla experiments. tion consensus (double underlined) followed by ATG. The Gene Cloning 65 reverse primers contained a restriction site (underlined) and a Partial sequences of the A12, A6, A5 desaturase and actin stop codon (double underlined). Following restriction and genes were obtained by PCR (Reddy Mix PCR Master Mix, ligation to the pYES2 vector (Invitrogen, Carlsbad, Calif., US 9,315,837 B2 35 36 USA), the constructs were used to transform S. cerevisiae transformed with YpPiELO1 by the PEG/lithium acetate strain W303 by the PEG/lithium acetate method R. D. Gietz, method. The yeast cells harboring the empty pYES2 vector R. A. Woods, Yeast Transformation by the LiAc/SS Carrier were used as control. Transformants were selected by uracil DNA/PEG Method, in: W. Xiao (Ed.), Yeast Protocols, Sec prototrophy on yeast synthetic medium (YSM) lacking uracil ond Edition, vol. 313, Methods in Molecular Biology, (Invitrogen, Carlsbad, Calif.). For functional expression, a Humana Press Inc, Totowa, N.J., 2006, pp. 107-120. The minimal selection medium containing 2% (w/v) raffinose was yeast cells harboring the empty pYES2 vector were used as inoculated with the YpPiELO1-transformants and grown at control. Transformants were selected by uracil prototrophy 27.degree. C. for 24h in a water bath shaker. Five ml of sterile on yeast synthetic medium (YSM) lacking uracil (Invitrogen, YSM, containing 1% (w/v) Tergitol-40 and 250.mu.M of the Carlsbad, Calif., USA). For functional expression, a minimal 10 appropriate fatty acid was inoculated with raffinose-grown selection medium containing 2% (w/v) raffinose was inocu cultures to obtain an OD of 0.2 at 600 nm. Expression was lated with the pYPiDesA12, pYPiDesA6 or pYPiDesA5 induced by adding galactose to a final concentration of 2% transformants and grown at 27.degree. C. for 24h in a water (w/v) and cultures were further grown at 27.degree. C. for 48 bath shaker. Five ml of sterile YSM, containing 1% (w/v) h. Cells were harvested by centrifugation, washed twice with Tergitol-40 and 250 mu.M of the appropriate fatty acid sub 15 0.1% NaHCO, freeze-dried and used for fatty acid analysis. strate was inoculated with raffinose-grown cultures to obtain Primer Design and Validation for PiELO1 (Elongase) an OD of 0.2 at 600 nm. Expression was induced by adding Real-Time Quantitative PCR primer pairs were designed galactose to a final concentration of 2% (w/v) and cultures for the PiELOJ and the house keeping gene 18S SSU rRNA were further grown at 27.degree. C. for 48 h. Cells were using the PrimerQuest tool (http://testidtdna.com/Scitools/ harvested by centrifugation, washed twice with 0.1% Applications/Primerquest/). Parameters were set for a primer NaHCO, freeze-dried and used for fatty acid analysis. length of 19 to 26 bp, primer melting temperature of 60.0+- Generation of 5' and 3' End Fragments of the Putative P 0.1.0...degree. C., and amplicon length of 90 to 150 bases. Incisa PUFA Elongase Primer pairs were validated using seven serial fifty-fold dilu To generate the full-length cDNA of the putative PUFA tions of cDNA samples and standard curves were plotted to elongase, 3'- and 5'-rapid amplification of the cDNA ends 25 test for linearity of the response. The primer pairs and primer (RACE) was performed using a BD SmartTM RACE cDNA concentrations with reaction efficiencies of 100+-0.10% Amplification Kit (BD Biosciences Clontech, Foster City, were chosen for quantitative RT-PCR analysis of relative Calif.) according to the manufacturer's manual. To amplify gene expression. The nucleotide sequences and characteris the 5'-end, the reverse gene-specific primers (GSP) 5’-CCCG tics of primers used for quantitative RT-PCR analysis are GCTGCTGCCATGCTTCTGTG (EL5R1) (SEQID NO: 29) 30 5'-TGGGGTAGGGAGAGTAGGCCCAAGT (EL5RN) presented in Table 2. (SEQ ID NO: 30) were designed using the Primer3 online software (http://frodo.wi.mit.edu). Based on the nucleotide TABLE 2 sequence of the obtained 5'-end fragment, two forward GSPs, Parameters of the primers used in RTQPCR 5'-GCCTACATGTCCTCTGCCGCCTGCTA (EL3R1) 35 reactions (SEQ ID NO: 31) and the nested, 5'-GCGGGACATGG Ampli- PCR GAGGGCTCATCTATACC (EL3R2) (SEQ ID NO: 32), Col. effici were constructed to amplify the 3'-end of the target gene. Forward primer size ency RACE PCR reactions were conducted using 5' and 3'-RACE Gene Reverse primer (bp) (%) 40 Ready cloNAs made from 1 ug total RNA of N-starved cells PiFELO1 AAGCTGTACGAGTTTGTGGATACGCT 95 92.3 with 50.times. BD Advantage 2 polymerase mix (Clontech (SEO ID NO : 35) (FWD) Laboratories Inc., Mountain View, Calif.). The PCR products GGATATGGAAGCGTGGTGGTAGA of the expected size were excised and purified from the gel (SEO ID NO : 36) (REV) (NucleoSpin Extract II purification kit, Machery-Nagel, 18S TGAAAGACGAACTTCTGCGAAAGCA 12 O 96.8 Duren, Germany) and ligated into a pCEMT-Easy vector 45 SSU (SEO ID NO : 37) (FWD) (Promega, Madison, Wis.). The full length cDNA corre rRNA AGTCGGCATCGTTTATGGTTGAGA sponding to the P incisa putative PUFA elongase (designated (SEO ID NO : 38) (REV) PiELO1) was assembled from the 5' and 3' RACE fragments and its ORF was further subcloned into a pYES2 vector (Invitrogen, Carlsbad, Calif.). 50 Calculation of Gene Transcript Levels Expression and Functional Characterization of PiELO1 The mean fold changes in gene expression were calculated cDNA (Elongase) in the Yeast Saccharomyces Cerevisiae according to the method using the average of threshold cycle The ORF encoding for PiELO1 was amplified using (Ct) values from triplicate cDNA-primer samples. The ACt Pful Jltra II fusion HSDNA polymerase (Stratagene, LaJolla, followed by the AACt was calculated from the average Ct Calif.) with the forward primer, 5'-AGGAATTCAAAATG 55 values of the target and the endogenous genes. The transcript GCATTGACGGCGGCCT (PUFAEL5RES1) (SEQ ID NO: abundance of the PiELO1 gene was normalized to the endog 33), containing a restriction site (underlined) and a yeast enous control 18S SSU rRNA gene. The fold-change in gene translation consensus followed by ATG (double underlined) expression was calculated using 2^* to find the expression and the reverse primer 5'-CATTCTAGATTACTG level of the target gene which was normalized to the endog CAGCTTTTGCTTGGCTGC (PUFAEL3RES2) (SEQ ID 60 enous gene, relative to the expression of the target gene at NO:34) containing a restriction site (underlined) and a stop time 0. codon (double underlined). The amplified sequence was then Fatty Acid Analysis restricted with EcoRI and Xbal (NEB, Ipswich, Mass.). The Fatty acid methyl esters (FAMES) were obtained by trans expected bands were gel-purified with NucleoSpin Extract II methylation of the freeze-dried P incisa or yeast biomass, purification kit (Machery-Nagel GmbH, Duren, Germany) 65 with dry methanol containing 2% H2SO4 (v/v) and heating at and ligated into a EcoRI-Xbal cut pYES2 vector, yielding 80.degree. C. for 1.5 h while stirring under an argon atmo YpPiELO1. Saccharomyces cerevisiae strain W303 was sphere. Gas chromatographic analysis of FAMES was per US 9,315,837 B2 37 38 formed on a Thermo Ultra Gas chromatograph (Thermo Sci TABLE 3 entific, Italy) equipped with PTV injector, FID detector and a fused silica capillary column (30 m. times. 0.32 mm; ZB Primers used in RTOPCR experiments WAXplus, Phenomenex). FAMES were identified by Forward primer/ Amplicon co-chromatography with authentic standards (Sigma Chemi 5 Gene Reverse primer size (bp) cal Co., St. Louis, Mo.; Larodan Fine Chemicals, Malmo, PiDes12 5 - GAAGCACCACCAAGGATGAGGT 112 Sweden) and FAME of fish oil (Larodan Fine Chemicals). (SEO ID NO: 39) (FWD) Each sample was analyzed in duplicates of three independent 5'-AGCGAGACGAAGATGACCAGGAA experiments. The structures of fatty acids were confirmed by (SEO BD NO: 4 O) (REV) 10 GC-MS of their pyrrolidine derivatives W. W. Christie, The PiDesó 5'-ACTTCCTGCACCACCAGGTCTTC 112 analysis offatty acids in: W.W. Christie (Ed.), Lipid analysis (SEO ID NO: 41) (FWD) Isolation, separation, identification and structural analysis of 5-TCGTGCTTGCTCTTCCACCAGT lipids, Vol. 15. Third edition, The Oily Press, Bridgewater, (SEO ID NO: 42) (REV) England, 2003, pp. 205-225 on HP 5890 equipped with a 15 PiDes5 5 - TAAGTGCCAGGGCTGTGCTAGA 11O mass selective detector (HP5971A) utilizing a HP-5 capillary (SEO ID NO : 43) (FWD) column and a linear temperature gradient from 120 to s' - GAACTGACCCTCCTCTGTGTCCT 300.degree. C. (SEO ID NO. 44) (REV) PiAct 5 - CGTCCAGCTCCACGATTGAGAAGA 154 Lipid Analysis (SEO ID NO: 45) (FWD) The biomass of S. cerevisiae was heated with isopropanol 5-ATGGAGTTGAAGGCGGTCTCGT at 80.degree. C. for 10 min and lipids were extracted by the (SEO ID NO: 46) (REV) method of Bligh-Dyer (1959) E. G. Bligh, W.J. Dyer, Arapid method of total lipid extraction and purification, Can. J. Bio Example 1 chem. Physiol. 37 (1959) 911-917. Total lipid extract was 25 separated into neutral and polar lipids by silica Bond-Elute cartridges (Varian, Calif.) using 1% of ethanol in chloroform Isolation and Identification of CDNAS for A12, A6, (V/v) and methanol to elute neutral and polar lipids, respec and A5 Desaturase Genes of P Incisa tively Z. Cohen, S. Didi, Y. M. Heimer, Overproduction of gamma.-linolenic and eicosapentaenoic acids by algae, 30 The partial sequences of the A12, A6 and A5 desaturase Plant Physiol. 98 (1992) 569-572). gene homologues were isolated using degenerate oligonucle otides (Table 1) targeting conserved amino acid motifs iden Polar lipids were separated into individual lipids by two tified in algae, lower plants and fungi. A partial sequence of dimensional TLC on Silica Gel 60 glass plates (10.times. 10 cm, 0.25 mm thickness (Merck, Darmstadt, Germany) the actin gene was amplified to be used as a house keeping 35 gene in RTOPCR experiments. according to Khozin et al. II. Khozin, D. Adlerstein, C. Partial sequences of 503, 558 and 636 bp, corresponding to Bigogno, Y. M. Heimer, Z. Cohen, Elucidation of the Biosyn the A12, A6, and A5 desaturase genes, respectively, were used thesis of Eicosapentaenoic Acid in the Microalga Porphy for designing gene specific primers that were used to amplify ridium cruentum (II. Studies with Radiolabeled Precursors), the 5’- and 3'-ends of the expected genes. Assembling the 5' Plant Physiol. 114 (1997) 223-230). Neutral lipids were 40 and 3' RACE PCR product sequences resulted in the identi resolved with petroleum ether:diethyl ether:acetic acid (70: fication of three cDNA clones with sequence homologies to 30:1, V/v/v). Lipids on TLC plates were visualized by brief known A12, A6, and A5 desaturase genes. The full-length exposure to iodine vapors, scraped from the plates and were cDNAS corresponding to A12, A6, and A5 desaturase genes transmethylated for the fatty acid analysis as previously were thus designated PiDes12, PiDes6, and PiDes5. The described. 45 ORFs for PiDes 12, PiDesG and PiDes5 genes were 1140, 1443, and 1446 by in length, respectively, coding for the Real-Time Quantitative PCR corresponding predicted proteins of 380, 481 and 482 amino Template cDNA for real-time quantitative PCR acids. The predicted amino acid sequence of PiDes12 is 64% (RTOPCR) was synthesized using 1 mu.g. of total RNA in a and 62% identical to that of Chlorella vulgaris (BAB78716) total volume of 20-mu.l., using oligo dT primer (Reverse 50 and Chlamydomonas reinhardtii (XP 001691669), respec iTTM 1 Strand Synthesis Kit, ABgene, Surrey, UK). Each tively, while it shares more than 50% identity with those of 20-mu.L. cDNA reaction was then diluted 3-fold with PCR higher plants. It contains three conserved histidine motifs grade water. HXXXH, HXXHII and HXXHH. The deduced amino acid Primer Design and Validation for Real-Time Quantitative sequence of PiDesG is 52% and 51% identical to those of the PCR 55 A6 desaturases from the liverwort M. polymorpha (AAT85661) and the moss Ceratodon purpureus, respec Real-Time Quantitative PCR (RTOPCR) primer pairs were tively (CAB94993). It is also 45% identical to the M. alpina designed for the PiDes 12, PiDesG, and PiDes5 genes and the A6 desaturase (ABN69090). PiDes5 shares 55 and 51% iden house keeping gene actin, PiAct using the PrimerQuest tool tity with the A5 desaturase from the microalgae M. squamata (http://testidtdna.com/Scitools/Applications/Primerquest/). 60 (CAQ30478), and O. tauri (CAL57370), respectively, and Primer pairs were validated as described by Iskandarov et al. 54% with that from M. polymorpha (AAT85663) but is only U. Iskandarov, I. Khozin-Goldberg, R. Ofir, Z. Cohen, Clon 36% identical to the M. alpina A5 desaturase (AAC72755). ing and Characterization of the A6 Polyunsaturated Fatty Both PiDesG and PiDes5 contain N-terminal fused cyto Acid Elongase from the Green Microalga Parietochloris chrome b5 domain including the HPGG motif and the three incisa, Lipids 44 (2009) 545-554). The nucleotide sequences 65 histidine boxes found to be conserved in front-end desatu of primer pairs and the amplicon sizes are presented in Table rases. The three characteristically conserved histidine-rich 3. motifs with amino acid patterns of HDexxH, HxxHH, US 9,315,837 B2 39 40 QXXHH in A6 desaturases, and HDXXH, QHXXXHH, QXXHH expression of PiDesG in the presence of 18:3c)3 resulted in the in A5 desaturases are also present in PiDesG and PiDes5, appearance of the corresponding A6 desaturation product respectively (FIG. 1). 18:4c)3 (FIG. 3). PiDesG desaturase was neither active on Phylogenetic Analysis endogenous yeast fatty acids nor on external 18:1. Piles.6 An unrooted phylogenetic tree (FIG. 2) of PiDes 12, 5 was not active on 20:36003 either, whereas PiDes5 desatu PiDesG. PiDes5 and several functionally characterized rated it to the non-methylene-interrupted 20:4,11,14.17. desaturases from all three groups were constructed to identify PiDes5 converted 20:4c03 into the respective A5 product, 20:5c)3 (EPA) as well as the added 18:1 into the non-meth their functional and phylogenetic relationships by the neigh ylene-interrupted 18:2^ (FIG. 3). The A5 position on bor joining program in MEGA4K. Tamura, J. Dudley, M. 18:2^ was determined by a characteristic peak of m/z-180 Nei, S. Kumar, MEGA4: Molecular evolutionary genetics 10 on the GC-MS spectra of its pyrrolidine derivative (not analysis (MEGA) software version 4.0, Mol. Biol. Evol. 24 shown). The presence of 18:2^' was also observed in the (2007) 1596-1599. The deduced amino acid sequence of chromatograms of the PiDes5 transformant supplied with PiDes12 is closely related to A12 desaturases of green algae C20 fatty acids. In addition, a tiny peak, tentatively identified and very similar to those of higher plants. The sequences of as 18:4'''''' was present on the chromatogram of the PiDes6 and PiDes5 cluster with A6 and A5 desaturases, 15 PiDes5 transformant fed with 18:30 (Table 4). respectively, from algae, moss and fungi. PiDes6 is highly similar to the M. polymorpha (MpDES6) and P tricornutum TABLE 4 (PtD6p) A6 desaturases, while PiDes5 appears to be closely related to the A5 desaturase from the moss M. polymorpha Conversion percent of the Supplied fatty acids by PiDesG and and shares more sequence similarity with the A5 desaturase PiDess in S. cerevisiae from the chlorophytes M. squamata and O. tauri than with Desaturase product those of fungal origin. However, both A6 or A5 desaturases Fatty acid and conversion (%)* from M. squamata and O. tauri appear to be structurally more similar to each other than to any of the known desaturases Substrate PiDesA6 PiDesA5 from either group. 25 18:1A9 18:2A59 (4.2) 18:249-12 18:2A6.9.12 (5.1) Example 2 18:349,12,15 18:4A69,12,15 (4.5) 18:4A5,9,12,158 (14) 18:3A6.9.12 20:3A11.14.17 20:4A5, 11.14.17 (10.0) Functional Expression of PiDes 12, PiDesG, and 20:3A8.11.14 20:4A5:8,11,14 (16.4) PiDess in S. Cerevisiae 30 204A8.11.14.17 20:5A5,8,11,14.17 (17.1) The functional activities of the proteins encoded by *calculated as the ratio of product(substrate + product) PiDes 12, PiDesG and PiDes5 were examined by heterologous tentative identification expression in S. cerevisiae. To this aim, the pYES2 constructs A kinetic analysis of ARA emergence was conducted in pYPiDes12, pYPiDesG and pYPiDes5 containing the ORFs 35 total fatty acids of the PiDes5 transformant during 24h fol for PiDes 12, PiDesG, and PiDes5, respectively, were trans lowing the addition of DGLA. Results showed that ARA peak formed into S. cerevisiae. GC analysis of the FAMEs of the was evident already after 3 h (corresponding to 10.9% sub yeast transformed with pYPiDes 12, revealed an appearance strate conversion) with a gradual but slow increase (up to of a small peak corresponding to 18:2 (0.3% of TFA; not 15.1% conversion) after 24h. Fatty acid analysis of the major shown). An attempt to improve the expression of the recom 40 polar and neutral lipids of the yeast transformed with pYP binant protein and to increase the activity by the modification iDes5 was performed 24 h after feeding with 20:3()6 to study of yeast translation consensus was not successful. The yeast the pattern of distribution of ARA within individual lipids. In cells harboring the empty vector, pYES2 (control) did not the transformed yeast, ARA appeared in all major phospho demonstrate desaturation activity on the added Substrates lipids (Table 4), with the highest proportion detected in PC. It (FIG. 3). 45 was also present in the neutral lipids, TAG, free fatty acids PiDesG and PiDes5 expressions were induced in the pres (FFA), diacylglycerol (DAG) and sterol esters (SE). Taking ence of the main ()6 substrates for A6 or A5 fatty acid desatu into account that PC, a major phospholipid of S. cerevisiae, rases, 18:2006 and 20:3 (D6, respectively. New peaks corre constituted for about 16% of total lipids (Table 5), it is obvi sponding to 18:3c)6 and 20:4c06, respectively, were detected, ous that PC allocated the main part of ARA attached to phos confirming the predicted function of PiDes6 and PiDes5. The pholipids. TABLE 5 Fatty acid composition and distribution of individual lipid classes of S. cerevisiae expressing the PiDess ORF. Fatty acid composition (% of total fatty acids % % O:406 Lipid 16:0 16:1 18:0 18:109 18:2^-) 20:306 20:406 conversion of TL* %TL

TAG 17.4 27.6 9.6 23.0 0.4 19.6 2.0 9.3 61.3 55.2 SE 1O.S. 38.5 7.1, 30.4 O.O 12.2 1.1 8.4 3.6 1.8 DAG 27.9 16.3 21.9 24.3 O.1 7.2 2.0 24.0 1.6 1.4 FFA 26.1 20.3 23.5 8.9 O.2 17.2 3.6 17.2 5.5 9.1 PC 19.7 30.1 9.3 24.3 0.7 11.6 3.6 24.2 15.7 25.7 PE 21.7 34.9 2.8 32.8 O.S S.O 1.6 24.6 3.2 2.3 PIPS 34.O 20.8 12.4 26.5 O.2 4.6 1.1 18.1 9.0 4.4

*TL total lipids US 9,315,837 B2 41 42 Example 3 PiELO1 deduced amino acid sequences was obtained using the algorithm available in the DAS transmembrane prediction Expression Profiles of PiDes 12, PiDesG, and PiDes5 server (http://www.sbc.su.se/..about.miklos/DAS/). The two Under Nitrogen Starvation strictly hydrophobic transmembrane domains were found about 50 amino acids downstream and upstream from the N To use actin as a house-keeping gene in quantitative real and C termini, respectively, while the two less hydrophobic time PCR experiments, a partial fragment (503 bp) of the P. domains were located about 100 amino acids downstream and incisa actin gene was amplified using the primers whose upstream from the N and C termini, respectively (FIG. 6). design was based on the C. reinhardtii actin cDNA (XM 001699016). Indeed, the expression level of the actin 10 Example 2 gene did not significantly change throughout the nitrogen starvation. PiDes 12, PiDesG, and PiDes5 were upregulated Phylogenetic Analysis following the transfer to Nitrogen starvation, reaching the highest expression level on day 3 and decreasing thereafter to An unrooted phylogenetic tree of the PiELO1 and several a level about 15 to 20 fold higher than that at time 0 (FIG. 4). 15 functionally characterized PUFA elongases was constructed Both the PiDes 12 and PiDes5 genes were expressed at levels to identify their functional and phylogenetic relationships by approximately 65 to 70 fold higher on day 3 than at time 0, the neighbor-joining program in MEGA4. According to FIG. while the PiDesG transcript was about 45 fold higher (FIG. 4). 7 one can see that PiELO1 falls into a group of PUFA elon The expression patterns of PiDes12, PiDesG, and PiDes5 gases of lower eukaryotes. Although the group contains correlated with the enhanced biosynthesis of ARA in P incisa mostly PUFA elongases with A6 activity, some A5 elongases, cells (Table 6). e.g., that of M. polymorpha and Leishmania infantum, are TABLE 6 Maior fatty acid composition of P incisa cells grown under N-Starvation Time Fatty acid composition (% of total fatty acids TFA (d) 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:306 18:303 20:306 20:466 20:5c)3 (%DW) O 19.1 5.6 4.1 2.9 3.1 9.1 20.1 1.2 6.O O.S 23.0 0.7 6.4 1.S 15.9 3.1 2.3 2.1 3.8 15.6 1S6 2.1 2.9 1.O 30.3 O6 8.7 3 12.7 2.3 1.5 1.9 3.8 15.2 13.5 1.6 2.0 O.9 39.7 O6 11.0 7 10.7 1.0 O6 1.1 3-5 14.9 1 O.O 1.1 O.9 1.O SO.O O.S 21.2 14 9.0 O2 O.3 O.8 3.1 13.4 8.8 O.9 O6 O.9 56.9 O6 29.0

Example 4 35 more related to A6 elongases of lower eukaryotes than to A5 elongases of higher eukaryotes. PiELO1 makes a closely Identification and Characterization of PiELO1 related subgroup with OtELO1, MpBLO1, MpBLO2 and PpELO1, the OtELO1 being the closest one. The BLASTX analysis (http://www.ncbi.nlm.nih.gov/ 40 Functional Expression of PiELO1 in S. Cerevisiae blast) of clones obtained through subtractive hybridization To characterize the enzymatic activity of PiELO1, the revealed a clone of 141 bp whose putative amino acid pYES2 plasmid containing the PiELO1 ORF downstream of sequence was highly homological to the C-terminal region of the GAL1 promoter was transformed into S. cerevisiae. The PUFA elongases. Using GSP primers, the 870 by 5'-end frag PiELO1 was expressed in the presence of the A6 PUFA elon ment was amplified and the sequence information was used to 45 gase substrates, 18:306 (gamma.-linolenic acid, GLA) and obtain the 3' end fragment from the 3' RACE Ready cDNA. 18:4c)3 (Stearidonic acid, STA). GC analysis of the FAMES Alignment of the 800 by 3'-end sequence with that of the of transformed yeast cells showed that PiELO1 elongated 5'-end fragment provided an overlapping nucleotide GLA and STA, converting them into dihomo-gamma.-li sequence and included the partial 141 bp sequence, thus noleic acid (DGLA, 20:3c)6) and eicosatetraenoic acid (20: confirming the amplification of both ends of the expected 50 4c03), respectively (FIG. 8). The yeast cells harboring the gene. The assembled complete 867 by cINA sequence, des empty vector alone did not demonstrate any elongation activ ignated as PiELO1, preceded and followed by 22 and 150 by ity on the added substrates, confirming that the PiELO1 nucleotides at 5' and 3' UTR, respectively. PiELO1 contained encoded enzyme has a A6 PUFA elongase activity. Feeding an ORF of 289 predicted amino acid residues consistent with the PiELO1 transformants with the Co6 fatty acids, LA and functionally characterized PUFA elongase ORFs from fungi, 55 ARA, did not result in their elongation (not shown). lower plants and algae (FIG. 5). The deduced amino acid Real-time quantitative PCR was performed to quantitate sequence of the PiELO1 was 50% identical to O. tauri and M. the alterations in expression levels of the A6 PiELO1 in P. polymorpha A6 PUFA elongase, while sharing 48 and 44% incisa cells under nitrogen starvation. The expression levels identity with P. patens A6 elongase and M. polymorpha A5 of the genes under nitrogen starvation were measured and elongase, respectively. The PiELO1 is also similar, yet with a 60 normalized to the expression level of the endogenous control lower score, to A6 elongases of fungal origin. It shares 40 and gene 18S SSU rRNA. The fold change in the expression level 36% identity with the A6 PUFA elongases of Thraus of the target genes in P incisa cells grown for 3, 7 and 14 don tochytrium and M. alpina (not included in the alignment), N-free medium was calculated relative to the expression level respectively. of the target genes in the log phase (time 0). The results The predicted amino acid sequence of the PiELO1 con 65 showed that during nitrogen starvation the mRNA of the tained four conserved motifs that are characteristic for PUFA PiELO1 gene was induced to its highest level at day 3 (14 fold elongases (FIG. 5, highlighted). The hydropathy plot of the increase over time 0), decreasing thereafter to a level still US 9,315,837 B2 43 44 higher than that of day 0 (FIG. 9). After 7 and 14 d, the inserted sequence. A similar low activity in yeast was also expression level of the PiELO1 gene was still 7.5 and 4.3 fold demonstrated in some cases, such as for A5 and A12 desatu higher, respectively. The level of expression of the PiELO1 rases of O. tauri and Chlorella vulgaris NJ-7, respectively. gene correlated with the increase in the share of ARA and the PiDesG and PiDes5 desaturated both ()3 and ()6 fatty acids C20/(C16+C18) elongation ratio (Table 7). The share of the with similar efficiency (FIG. 3; Table 4). Various results con elongation product, DGLA, increased sharply at day 3 (50% cerning ()3/()6 substrate preference were reported for func increase over time 0) and decreased thereafter. tionally characterized A6 and A5 desaturases from different TABLE 7 Major fatty acid composition of F incisa cells grown under N-starvation Time Fatty acid composition (% of total fatty acids Elo. (days) 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:306 18:303 20:306 20:46)6 20:5c)3 ratio O 19.1 S6 4.1 2.9 3.1 9.1 20.1 1.2 6.O O.S 23.0 0.7 O.34 3 12.7 2.3 1.5 1.9 3.8 15.2 13.5 1.6 2.0 O.9 39.7 O6 O.74 7 10.7 1.0 O.6 1.1 3-5 14.9 10.O 1.1 O.9 1.O SO.O O.S 1.10 14 9.0 O2 O.3 O.8 3.1 13.4 8.8 O.9 O6 O.9 56.9 O6 1.44 Elongation ratio, C20 (C18 + C16) 2O The capacity of P incisa to accumulate large quantities of organisms that were expressed in yeast. A front-end PiDes5 ARA-rich TAG under nitrogen starvation, Suggested that it desaturated its principal substrate 20:3 (D6 as well as 20:4c)3; would be of great interest to study its genes and enzymes in addition, non-methylene interrupted fatty acids were also involved in the accumulation of VLC-PUFA. In the present 25 produced as a result of its activity on 20:3c)3, and on both work, P incisa A12, A6, and A5 desaturases were cloned and endogenous and exogenous 18:1, but with lower efficiency. studied, which in conjunction with a recently cloned A6 spe PiDes5 produced 18:2.^ from 18:1 but was more active cific PUFA elongase U. Iskandarov, I. Khozin-Goldberg, R. when 18:1 was exogenously supplied. CrDES did insert A5 Ofir, Z. Cohen, Cloning and Characterization of the A6 Poly double bond on both 18:1 and 18:2 producing the non-meth unsaturated Fatty Acid Elongase from the Green Microalga 30 ylene interrupted 18:2^' and 18:3^'', while adding a A7 Parietochloris incisa, Lipids 44 (2009) 545-554.), representa double bond to 20:2a)6 and 20:30a). Apparently, in addition to set of P incisa genes involved in the biosynthesis of ARA.U. the fatty acid chain length, the location and number of double Iskandarov et al., 2009 is incorporated by reference as if fully bond, and the form of the substrate (lipid- or CoA bound) are set forth herein. also crucial for A5 desaturation. The his-boxes of A12, A6 and A5 desaturases including 35 PiDesG desaturated neither the yeast major monounsat PiDes 12, PiDesG, and PiDes5 are detailed in Table 8. urated fatty acids nor the exogenously supplied 18:1. Piles.6 did not act on 20:3c)3, indicating that it is specific for A9 C18 TABLE 8 PUFA. It appears that not only the organisms being trans Conserved histidine rich motifs of A12, A6, and A5 desaturases formed, but also the gene origin, determine the Substrate 40 specificity of the recombinant A6 and A5 desaturase. Func Des A12 HECxH HxxHH HxxHH tional characterization of PiDes6 and PiDes5 confirmed the Des A6 HDXXH HxxHH QxxHH previously reported substrate specificities of these desatu Des AS HDxxH QHxxxHH QxxHH rases which were generally restricted to C18 and C20 sub strates, respectively. Notably, cysteine (C) in the first his-box and the first his 45 In P incisa it was shown that PC and DGTS are used for tidine (H) in the third his-boxes, respectively, are conserved lipid-linked C18 A6 desaturation whereas mostly PE is used only in A12 desaturases. The second residue in the first his for C20 A5 desaturation. PiDes5 featured higher substrate box of the all three types of desaturases is acidic; in A6 and A5 conversion rate in comparison to PiDesG. A relatively fast desaturases it is mostly aspartic acid (D), and in A12 desatu emergence of substantial percentage of ARA (10.6% conver rases mostly glutamic acid (E). This indicates the importance 50 sion after 3 hoffeeding) pursued us to study ARA distribution of an acidic residue at this position for desaturation. Similarly in individual lipid classes of the transformed yeast (24 h of to other A6 and A5 desaturases glutamine (Q) is found in the feeding). ARA was detected in all analyzed phospho- and third his-box of PiDesG and PiDes5 (FIG. 1: Table 8) and in neutral lipid classes of S. cerevisiae expressing PiDes5 (Table the second his-box of A5 desaturases. The replacement of the 5). Similar conversion percentages were determined in all H residue with Q in the third his-box of A6 and A5 desaturases 55 analyzed phospholipids, however, ARA distribution showed points to the role of Q in PUFA desaturation. Indeed, replac preference for the major phospholipids, PC, followed by PE. ing this Q with histidine or isoleucine eliminated the enzyme In P incisa, PE was found to be the main site for lipid-linked activity of the recombinant A6 desaturase in yeast cells. A5 desaturation C. Bigogno, I. Khozin-Goldberg, D. Adler Glutamine was also found to be highly conserved in the third stein, Z. Cohen, Biosynthesis of arachidonic acid in the ole his-box of the A4 desaturases Pavlova lutheri (AY332747), 60 aginous microalga Parietochloris incisa (Chlorophyceae): Euglena gracilis (AY278558), and Thraustochytrium sp. Radiolabeling studies, Lipids 37 (2002) 209-216), while PC, (AF489589). a major A6 acyl lipid desaturation Substrate in this organism, Heterologous expression of PiDesG and PiDes5 in yeast was assumed to be utilized for A5 desaturation, too. cells confirmed their A6 and A5 activity by conversion of The quantitative RT-PCR results revealed that the gene Supplemented fatty acids to the corresponding desaturation 65 expression levels of PiDes12, PiDesG, and PiDes5 followed a products. PiDes 12 demonstrated very low desaturation activ similar pattern during the course of nitrogen starvation. The ity, which could not be enhanced by the 5' modification of the major transcriptional activation of the all three desaturases US 9,315,837 B2 45 46 occurred on day 3 coinciding with a sharp rise in the percent PiELO1 is another example of a single step A6 PUFA age of ARA, which almost doubled (FIG. 4, Table 6). The elongases cloned from an algal species. Similarly to GLELO of M. alpina, PiELO1 prefers the A6 C18 PUFA substrates, same expression pattern featured the P incisa A6 PUFA elon GLA and STA. Only these A6 fatty acids were, when exog gase, however, at lower level. It was shown in radiolabeling enously added, elongated to the respective products by S. and inhibitor studies, that ARA biosynthesis in P incisa fol cerevisiae cells transformed with PiELO1 (FIG. 8). Transfor lows the ()6 pathway. The concerted transcriptional activation mation of a higher plant so as to render it to produce A6 PUFA of the PiDes12, PiDesG, PiDes5 and PiELO1 genes was requires that the elongase used will have a high selectivity for accompanied by an increase in the percent share of 18:1, a A6 PUFA, thereby reducing the appearance of side products main chloroplast-derived fatty acid exported to ER and sub in the transformed plant. Bifunctional invertebrate PUFA 10 elongases with both A6 and A5 activities (OmELO, XiELO, strate of A12 desaturase. High expression of ARA biosyn and CiELO) are less desirable in plant transformations. thetic genes was accompanied by enhanced A5 and A6 desatu Phylogenetic analysis showed (FIG. 6) that the PUFA elon rations (Table 6). gases are not strictly divided according to their substrate In conclusion, the A12, A6 and A5 fatty acid desaturases specificity. The A6 elongases of algal (OtBLO1, TpELO1, involved in ARA biosynthesis in P incisa were identified and PiELO1) and moss (PpELO1) origin are functionally functionally characterized. The corresponding ORFs 15 restricted to the elongation of A6-C18-PUFAs, however these PiDes 12, PiDesG, and PiDes5, expressed in yeast confirmed elongases are placed in separate groups on the phylogenetic their favorable enzymatic activity. Nitrogen starvation led to tree (FIG. 7). PiELO1 is closely related to OtBLO1 isolated an increased transcription of the cloned genes reaching maxi from a chloropyte and a lowerplant rather than to ELO1 genes mum on day 3 and enhanced accumulation of ARA thereafter. isolated from a diatom, although both are specific for the Understanding the mechanisms underlying gene transcrip elongation of A6-C18-PUFAs (FIG. 7). PiELO1 is highly tion regulation in metabolic pathways and characteristics of similar to and is placed in the same group with both A6 and A5 enzymes involved in ARA and lipid biosynthesis in P incisa elongases of the liverwort M. polymorpha. Kajikawa et al. is a prerequisite for manipulating algal species to produce Suggested that MpELO2, a A5 elongase, is likely to have Sustainable oils of pharmaceutical and nutraceutical values. originated through gene duplication of the MpELO1 gene. A cDNA (PiELOJ) of an elongase encoding for a deduced 25 The algal A5 PUFA elongases, OtBLO2, TpELO2 and the P protein was isolated from P incisa, structurally similar to A6 salina ELO1 are more likely to share a common branch with PUFA elongase gene products from microalgae, lower plants the mammalian and animal A5 PUFA elongases, OmELO and and fungi (FIG. 5). The deduced amino acid sequence of the HsELO2, while they are also similar to bifunctional PUFA PiEIO1 ORF was about 50% identical to that of A6 elongases elongases such as CiELO 1/2. from the liverwort M. polymorphs (AAT85662), the green 30 Quantitative real timed PCR studies revealed that the marine microalga O. tauri (AAV67797) and the moss P. pat expression level of the PiELO1 gene was up regulated during ens (AAL84174). In similarity to recently cloned PUFA elon the time course of N-starvation (FIG.9). Nitrogen starvation gases, the predicted protein is highly hydrophobic and has led to a continuous increase in the share of ARA and the two strongly hydrophobic transmembrane regions, the first C20/(C16+C18) elongation ratio (Table 7). However, a major one located about 50 amino acids downstream of the N-ter transcriptional activation of PiELO1 which occurred on day 3 minus and the second one in the vicinity of the C-terminus. 35 (14-fold increase in transcript level) coincided with the steep The PiELO1 sequence was identified in a C-terminal lysine rise in AA accumulation and elongation ratio (Table 7). The rich motif, important for the endoplannic reticulum targeting, increase in PiELO1 transcription level followed by enhanced as well as four conserved motifs FYXSKXXEFxDT (SEQ ID biosynthesis of ARA may be interpreted as an increase in NO: 62), QxxxLHVYHHxxI (SEQ ID NO: 63), NSXXH PiELO1 enzyme level and/or enzymatic activity. The impor VxMYXYY (SEQID NO: 64) and TxxOxxQF (SEQID NO: 40 tance of the transcriptional activation of PiELO1 is supported 65), including a highly conserved histidine box suggested to by the fact that PUFA elongase was the only ARA biosynthe be functionally important for PUFA elongation (FIG. 5). sis related gene that was obtained from the subtractive library. These conserved motifs were not found in other classes of The significance of the coordinated transcription and plant microsomal elongases, 0 ketoacyl CoA synthases and action of desaturases and elongases in ARA biosynthesis in P fatty acid elongases (FAE) involved in extraplastidial elonga 45 incisa is yet to be elucidated. Possibly, the elongation of GLA tion of Saturated and monounsaturated fatty acids. A variant by PiELO1 could be rate-limiting in ARA biosynthesis as it is histidine box QAFHH with three replacements in C18-A9 in M. alpina. Abbadi et al. (2004) speculated that in trans PUFA elongase IgASE1 from I. galbana is thought to be genic plants modified with VLC-PUFA biosynthesis genes, important for enzymatic activity rather then for substrate substrate availability rather than enzymatic activity is rate specificity. limiting in the A6 elongation of PUFA.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 65

<21 Oc SEO ID NO 1 <211 LENGTH: 379 <212> TYPE PRT ORGANISM: Parietochloris incisa

<4 OOs SEQUENCE: 1 Met Gly Lys Gly Gly Cys Tyr Glin Ala Gly Pro Pro Ser Ala Lys Llys 1. 5 1O 15

Trp Glu Ser Arg Val Pro Thr Ala Lys Pro Glu Phe Thr Ile Gly Thr 2O 25 3 O US 9,315,837 B2 47 48 - Continued

Lell Arg Lys Ala Ile Pro Wall His Phe Glu Arg Ser Ile Pro Arg 35 4 O 45

Ser Phe Ala Lell Ala Ala Asp Luell Ala Ala Ile Ala Wall Met Tyr SO 55 6 O

Tyr Luell Ser Thir Phe Ile Asp His Pro Ala Wall Pro Arg Wall Luell Ala 65 70

Trp Gly Luell Luell Trp Pro Ala Trp Tyr Phe Glin Gly Ala Wall Ala 85 90 95

Thir Gly Wall Trp Wall Ile Ala His Glu Cys Gly His Glin Ala Phe Ser 105 11 O

Pro Glin Trp Lell Asn Asp Ala Wall Gly Luell Wall Lell His Ser Cys 115 12 O 125

Lell Luell Wall Pro Tyr Ser Trp His Ser His Arg Arg His His 13 O 135 14 O

Ser Asn Thir Gly Ser Thir Thir Asp Glu Wall Phe Wall Pro Arg Glu 145 150 155 160

Ala Ala Met Wall Glu Ser Asp Phe Ser Luell Met Glin Thir Ala Pro Ala 1.65 17O 17s

Arg Phe Luell Wall Ile Phe Wall Ser Luell Thir Ala Gly Trp Pro Ala Tyr 18O 185 19 O

Lell Phe Ala Asn Ala Ser Gly Arg Gly Trp Ala Asn His 195

Phe Asp Pro Ser Pro Ile Phe Thir Arg Glu Arg Ser Glu Ile 21 O 215 22O

Wall Wall Ser Asp Wall Ala Lieu Thr Wall Wall Ile Ala Gly Luell Ser 225 23 O 235 24 O

Lell Gly Ala Phe Gly Trp Ala Trp Luell Wall Glu Wall Ile 245 250 255

Pro Luell Ile Wall Asn Met Trp Luell Wall Met Ile Thir Luell Luell Glin 26 O 265 27 O

His Thir His Pro Glu Lell Pro His Ala Asp Glu Trp Asp Trp 27s 28O 285

Lell Arg Gly Ala Lell Ala Thir Asp Arg Ser Tyr Gly Gly Met Pro 29 O 295 3 OO

Asp His Luell His His His Ile Ala Asp Thir His Wall Ala His His Luell 3. OS 310 315

Phe Ser Thir Met Pro His His Ala Glin Glu Ala Thir Glu Ala Ile 3.25 330 335

Pro Ile Luell Gly Lys Glin Asp Arg Asn Wall Trp 34 O 345 35. O

Ala Ala Luell Trp Glu Asp Phe Ser Luell Cys Arg Wall Ala Pro Asp 355 360 365

Thir Ala Gly Ser Gly Ile Lell Trp Phe Arg Ala 37 O 375

<210s, SEQ ID NO 2 &211s LENGTH: 212. TYPE : PRT <213> ORGANISM: Parietochloris incisa

<4 OOs, SEQUENCE: 2 Met Cys Glin Gly Glin Ala Val Glin Gly Lieu. Arg Arg Arg Ser Ser Phe 1. 5 15

Lieu Lys Lieu. Thr Gly Asp Ala Ile Lys Gly Ala Val Ala Ala Ile Ser 25 US 9,315,837 B2 49 50 - Continued

Asp Phe Asn Lell Pro Ala Ala Thir Pro Wall Phe Ala Arg Arg Ser 35 4 O 45

Lell Ser Asp Ser Ala Lell Glin Glin Arg Asp Gly Pro Arg Ser Lys Glin SO 55 6 O

Glin Wall Thir Luell Glu Glu Lell Ala Glin His ASn Thir Pro Glu Asp Cys 65 70 7s

Trp Luell Wall Ile Lys Asn Wall Asp Wall Ser Gly Trp Gly Pro 85 90 95

Glin His Pro Gly Gly His Wall Ile Tyr Thir Ala Gly Lys Asp Ala 105 11 O

Thir Asp Wall Phe Ala Phe His Ala Glin Thir Thir Trp Ser Glin Luell 115 12 O 125

Arg Pro Phe Ile Gly Asp Ile Wall Glu Glu Glu Pro Met Pro Ala 13 O 135 14 O

Lell Luell Asp Phe Arg Glu Luell Arg Thir Arg Lell Glin Glin Glin Gly 145 150 155 160

Lell Phe Arg Ser Asn Lell Luell Wall Ala Ser Thir 1.65 17O 17s

Lell Ser Luell Luell Ala Ala Ala Luell Ala Wall Luell Ile Thir Glin Arg Asp 18O 185 19 O

Ser Trp Luell Gly Lell Wall Gly Gly Ala Phe Luell Lell Gly Luell Phe Trp 195

Glin Glin Ser Gly Trp Lell Ala His Asp Phe Luell His His Glin Wall Phe 21 O 215 22O

Thir Asp Arg Glin Trp Asn Asn Wall Met Gly Tyr Phe Lieu Gly Asn Wall 225 23 O 235 24 O

Glin Gly Phe Ser Thir Asp Trp Trp Lys Ser His Asn Wall His 245 250 255

His Ala Wall Pro Asn Glu Lell Asp Ser Asp Ser Ala Ala Arg Asp 26 O 265 27 O

Pro Asp Ile Asp Thir Lell Pro Luell Luell Ala Trp Ser Ser Glu Met Luell 285

Asp Ser Met Ser Asn Ser Gly Ala Arg Luell Phe Wall Arg Met Glin His 29 O 295 3 OO

Tyr Phe Phe Phe Pro Ile Lell Luell Phe Ala Arg Met Ser Trp Glin 3. OS 310 315

Glin Ser Wall Ala His Ala Ser Asp Luell Ser Arg Thir Ser Ala Gly 3.25 330 335

Wall Glu Luell Ala Lell Ala Luell His Ala Trp Phe Luell Gly 34 O 345 35. O

Ala Ala Phe Ser Wall Lell Pro Pro Luell Lys Ala Wall Wall Phe Ala Luell 355 360 365

Lell Ser Glin Met Phe Ser Gly Phe Luell Luell Ser Ile Wall Phe Wall Glin 37 O 375

Ser His Asn Gly Met Glu Wall Ser Asp Thir Asp Phe Wall Thir 385 390 395 4 OO

Ala Glin Ile Wall Ser Thir Arg Ile Luell Ser Asn Wall Trp Asn Asp 4 OS 415

Trp Phe Thir Gly Gly Lell Asn Glin Ile Glu His His Luell Phe Pro 425 43 O

Thir Luell Pro Arg His Asn Lell Gly Wall Glin Ser Ile Met Glu 435 44 O 445 US 9,315,837 B2 51 - Continued Lieu. Cys His Llys His Gly Lieu Val Tyr Glu Asn. Cys Gly Met Ala Thr 450 45.5 460 Gly. Thir Tyr Arg Val Lieu. Glin Arg Lieu Ala Asn Val Ala Ala Glu Ala 465 470 47s 48O

<210s, SEQ ID NO 3 &211s LENGTH: 481 212. TYPE: PRT <213> ORGANISM: Parietochloris incisa

<4 OOs, SEQUENCE: 3 Met Met Ala Val Thr Glu Gly Ala Gly Gly Val Thr Ala Glu Val Gly 1. 5 1O 15 Lieu. His Lys Arg Ser Ser Glin Pro Arg Pro Ala Ala Pro Arg Ser Lys 2O 25 3O Lieu. Phe Thr Lieu. Asp Glu Val Ala Lys His Asp Ser Pro Thir Asp Cys 35 4 O 45 Trp Val Val Ile Arg Arg Arg Val Tyr Asp Val Thr Ala Trp Val Pro SO 55 6 O Glin His Pro Gly Gly Asn Lieu. Ile Phe Wall Lys Ala Gly Arg Asp Cys 65 70 7s 8O Thr Glin Lieu. Phe Asp Ser Tyr His Pro Lieu. Ser Ala Arg Ala Val Lieu. 85 90 95 Asp Llys Phe Tyr Ile Gly Glu Val Asp Val Arg Pro Gly Asp Glu Glin 1OO 105 11 O Phe Leu Val Ala Phe Glu Glu Asp Thr Glu Glu Gly Glin Phe Tyr Thr 115 120 125 Val Lieu Lys Lys Arg Val Glu Lys Tyr Phe Arg Glu Asn Llys Lieu. Asn 13 O 135 14 O Pro Arg Ala Thr Gly Ala Met Tyr Ala Lys Ser Lieu. Thir Ile Lieu Ala 145 150 155 160 Gly Leu Ala Leu Ser Phe Tyr Gly Thr Phe Phe Ala Phe Ser Ser Ala 1.65 17O 17s Pro Ala Ser Lieu Lleu Ser Ala Val Lieu. Lieu. Gly Ile Cys Met Ala Glu 18O 185 19 O Val Gly Val Ser Ile Met His Asp Ala Asn His Gly Ala Phe Ala Arg 195 2OO 2O5 Asn. Thir Trp Ala Ser His Ala Lieu. Gly Ala Thr Lieu. Asp Ile Val Gly 21 O 215 22O Ala Ser Ser Phe Met Trp Arg Glin Gln His Val Val Gly His His Ala 225 23 O 235 24 O Tyr Thr Asn. Wall Asp Gly Glin Asp Pro Asp Lieu. Arg Val Lys Asp Pro 245 250 255 Asp Val Arg Arg Val Thr Llys Phe Glin Pro Glin Glin Ser Tyr Glin Ala 26 O 265 27 O Tyr Glin His Ile Tyr Lieu Ala Phe Lieu. Tyr Gly Lieu. Lieu Ala Ile Llys 27s 28O 285

Ser Val Lieu. Lieu. Asp Asp Phe Met Ala Lieu. Ser Ser Gly Ala Ile Gly 29 O 295 3 OO

Ser Val Llys Val Ala Lys Lieu. Thr Pro Gly Glu Lys Lieu Val Phe Trp 3. OS 310 315 32O

Gly Gly Lys Ala Lieu. Trp Lieu. Gly Tyr Phe Val Lieu. Lieu Pro Val Val 3.25 330 335 Llys Ser Arg His Ser Trp Pro Lieu. Lieu Ala Ala Cys Trp Lieu. Lieu. Ser 34 O 345 35. O

US 9,315,837 B2 67 68 - Continued

<4 OOs, SEQUENCE: 35 actitcc tigca ccaccagg to titc 23

<210s, SEQ ID NO 36 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Parietochloris incisa

<4 OOs, SEQUENCE: 36 tcqtgcttgc tict tccacca git 22

<210s, SEQ ID NO 37 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Parietochloris incisa

<4 OO > SEQUENCE: 37 taagtgc.cag ggctgtgcta ga 22

<210s, SEQ ID NO 38 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Parietochloris incisa

<4 OOs, SEQUENCE: 38 gaactgaccc ticcitctgttgt cct 23

<210s, SEQ ID NO 39 <211 LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Parietochloris incisa

<4 OOs, SEQUENCE: 39 cgtc.ca.gctic cacgattgag aaga 24

<210s, SEQ ID NO 4 O &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Parietochloris incisa

<4 OOs, SEQUENCE: 4 O atggagttga aggcggtctic gt 22

<210s, SEQ ID NO 41 &211s LENGTH: 376 212. TYPE: PRT <213> ORGANISM: Chlorella vulgaris <4 OOs, SEQUENCE: 41 Met Ala Ala Thr Arg Arg Ala Pro Ser Ala Glu Gly Trp Thir Arg Glin 1. 5 1O 15

Pro Val Asn Thr Lys Pro Ala Phe Ser Val Ser Thir Lell Arg Ala 2O 25 3O

Ile Pro Ala His Cys Trp Glin Arg Ser Lieu. Pro Arg Ser Ala Tyr 35 4 O 45

Lieu Ala Ala Asp Lieu. Lieu Ala Lieu Ala Ala Luell Wall Trp Ala Ser Thir SO 55 6 O

Phe Ile Asp Ala Ala Pro Val Pro Ala Ala Wall Arg Trp Luell Ala Luell 65 70 8O

Trp Pro Ala Tyr Trp Tyr Lieu Ala Gly Ala Wall Ala Thir Gly Ile Trp 85 90 95 US 9,315,837 B2 69 70 - Continued

Wall Ile Ala His Glu Gly His Glin Ala Phe Ser Asp Tyr Glin Ala 1OO 105 11 O

Wall Asn Asp Gly Wall Gly Lell Wall Luell His Ser Lell Lell Luell Wall Pro 115 12 O 125

Tyr Ser Trp Lys His Ser His Arg Arg His His Ser Asn Thir Gly 13 O 135 14 O

Asn Wall Wall Asp Glu Wall Phe Wall Pro Pro Thir Arg Glu Glu Wall 145 150 155 160

Ser Asp Trp Glu Lell Glu Glin Ala Trp Pro Ile Arg Luell Wall Lys 1.65 17O 17s

Lell Phe Ile Thir Lell Thir Lell Gly Trp Pro Luell Lell Ala Phe Asn 18O 185 19 O

Wall Ala Ser Arg Pro Glu Lys Ser Trp Wall Asn His Phe Asp Pro 195

Trp Ser Pro Ile Phe Ser Lys Arg Glu Luell Wall Glu Wall Ala Wall Ser 21 O 215

Asp Ala Ala Luell Wall Ala Wall Luell Gly Luell Arg Glin Luell Ala Ala 225 23 O 235 24 O

Ser Phe Gly Trp Ala Trp Lell Wall Thir Trp Lell Wall Pro Tyr Luell 245 250 255

Wall Wall Asn Phe Trp Lell Wall Thir Ile Thir Met Lell Glin His Ser His 26 O 265 27 O

Pro Glu Luell Pro His Gly Glu Asp Glu Trp Asp Trp Luell Arg Gly 28O 285

Ala Lieu Thr Thr Wall Asp Arg Asp Gly Trp Lieu Lieu Asn Ser Lieu 29 O 295 3 OO

His His His Ile Ala Asp Thir His Wall Ala His His Lell Phe Ser Glin 3. OS 310 315

Met Pro His His Ala Glin Glu Ala Thir Glu Ala Lell Pro Wall 3.25 330 335

Lell Gly Asp Tyr Tyr Arg Ser Asp Ser Arg Pro Lell Lell Glin Ala Ile 34 O 345 35. O

Trp Glin Asp Phe Gly Ser Arg Wall Ala Pro Asp Thir Pro Gly 355 360 365

Asp Gly Wall Luell Trp Phe Arg 37 O 375

<210s, SEQ ID NO 42 &211s LENGTH: 383 212. TYPE : PRT &213s ORGANISM: Chlamydomonas reinhardtii

<4 OOs, SEQUENCE: 42

Met Thir Wall. Thir Arg Arg Gly Wall Asn Ile Glin Ala Asp Ala Thir 1. 5 1O 15

Asp Ser Ala Gly Glu Glin Arg Tyr Pro Ala Ala Pro Pro Thir Phe 25

Ser Luell Gly Asp Ile Arg Ala Ile Pro Ala His Cys Phe Glu Lys 35 4 O 45

Ser Ala Luell Arg Ser Phe Ala His Luell Ala Wall Asp Wall Thir Wall Cys SO 55 6 O

Ala Trp Luell Trp Tyr Gly Ser Thir Phe Ile Asp His Pro Ala Wall Pro 65 70

Arg Luell Ala Trp Phe Wall Luell Trp Pro Luell Trp Phe Trp Glin 85 90 95 US 9,315,837 B2 71 72 - Continued

Gly Ala Phe Met Thir Gly Ile Trp Wall Ile Ala His Glu Cys Gly His 105 11 O

Gly Ala Phe Ser Asn Ser Glu Ala Luell Asn Asp Gly Wall Gly Luell Wall 115 12 O 125

Met His Ser Luell Lell Lell Wall Pro Ser Trp His Ser His 13 O 135 14 O

Arg Arg His His Glin Asn Thir Gly Ser Thir Ala Lys Asp Glu Wall Phe 145 150 155 160

Wall Pro Ala Wall Lys Pro Ala Gly Thir Lys Ala Pro Trp His Arg 1.65 17s

Asn Pro Wall Tyr Arg Lell Gly His Ile Luell Phe Glin Glin Luell Luell Gly 18O 185 19 O

Trp Pro Luell Lell Lell Phe Asn Wall Ser Gly His Glu Pro Arg 195

Trp Ala Asn His Phe Asp Pro Phe Ser Pro Ile Phe Thir Arg Glu 21 O 215 22O

Arg Ile Glu Wall Lell Wall Ser Asp Ile Ala Luell Ala Wall Wall Wall Ala 225 23 O 235 24 O

Gly Luell Ala Ala Ile Ser Arg Thir Trp Gly Phe Met Phe Luell Luell 245 250 255

Thir Luell Ile Pro Lell Wall Wall Asn His Trp Lell Wall Met Ile 26 O 265 27 O

Thir Phe Luell Glin His Thir His Pro Luell Pro His Tyr Gly Asp Gly 28O 285

Glu Trp Asp Trp Lieu Arg Gly Ala Met Ala Thr Wall Asp Arg Ser 29 O 295 3 OO

Gly Wall Luell Asp His Wall Phe His His Ile Ala Asp Thir His Wall Ala 3. OS 310 315 32O

His His Luell Phe Ser Met Pro His Tyr His Ala Glu Glu Ala Thir 3.25 330 335

Glu Ala Ile Lys Lys Wall Lell Gly Asp Ala Asp Ser Arg 34 O 345 35. O

Asn Wall Phe Arg Ala Lell Trp Asp Glu Wall Gly Cys Ala Wall Wall 355 360 365

Ala Pro Asp Thir Asn Gly Pro Glu Glin Wall Trp His Arg 37 O 375 38O

<210s, SEQ ID NO 43 &211s LENGTH: 384 212. TYPE : PRT &213s ORGANISM: Gossypium hirsutum

<4 OOs, SEQUENCE: 43

Met Gly Ala Gly Gly Arg Met Ser Wall Pro Pro Ser Glin Arg Lys Glin 1. 5 1O 15

Glu Ser Gly Ser Met Arg Wall Pro Ile Ser Pro Pro Phe Thir 25

Lell Ser Glu Ile Lys Ala Ile Pro Pro His Phe Glin Arg Ser 35 4 O 45

Lell Ile Arg Ser Phe Ser Tyr Luell Wall Tyr Asp Phe Ile Luell Wall Ser SO 55 6 O

Ile Phe Tyr Wall Ala Thir Thir Phe His Asn Lell Pro Glin Pro 65 70

Lell Ser Phe Wall Ala Trp Pro Ile Trp Thir Lell Glin Gly Ser Wall 85 90 95 US 9,315,837 B2 73 74 - Continued

Lell Thir Gly Wall Trp Wall Ile Ala His Glu Cys Gly His His Ala Phe 105 11 O

Ser Asp Tyr Glin Trp Ile Asp Asp Thir Wall Gly Lell Ile Luell His Ser 115 12 O 125

Ser Luell Luell Wall Pro Phe Ser Trp Ser His Arg Arg His 13 O 135 14 O

His Ser Asn Thir Gly Ser Lell Glu Arg Asp Glu Wall Phe Wall Pro Lys 145 150 155 160

Arg Ser Ser Ile Arg Trp Trp Ala Lys Lell Asn Asn Pro Pro 1.65 17s

Arg Phe Wall Thir Wall Thir Ile Glin Luell Thir Lell Gly Trp Pro Luell 18O 185 19 O

Luell Ala Phe Asn Wall Ala Gly Arg Pro Glu Gly Luell Ala 195

His Tyr Asn Pro Tyr Gly Pro Ile Asn Asp Arg Glu Arg Luell Glin 21 O 215

Ile Ile Ser Asp Wall Gly Wall Luell Ala Wall Thir Gly Luell Tyr 225 23 O 235 24 O

Arg Luell Wall Luell Ala Gly Luell Ala Trp Wall Ile Wall Tyr Gly 245 250 255

Wall Pro Luell Luell Ile Wall Asn Ala Phe Luell Wall Met Ile Thir Luell 26 O 265 27 O

Glin His Thir His Pro Ala Lell Pro His Asp Ser Ser Glu Trp Asp 27s 28O 285

Trp Lieu Arg Gly Ala Lieu Ala Thr Wall Asp Arg Asp Gly Ile Lieu 29 O 295 3 OO

Asn Wall Phe His Asn Ile Thir Asp Thir His Wall Ala His His Luell 3. OS 310 315

Phe Ser Thir Met Pro His His Ala Met Glu Ala Thir Ala Ile 3.25 330 335

Pro Ile Luell Gly Glu Tyr Ser Phe Asp Gly Thir Pro Wall Tyr 34 O 345 35. O

Ala Ile Phe Glu Ala Lys Glu Ile Wall Glu Pro Asp 355 360 365

Glu Gly Glu Glin Ser Ser Lys Gly Wall Phe Trp Phe Arg Asn Ile 37 O 375 38O

<210s, SEQ ID NO 44 &211s LENGTH: 381 212. TYPE : PRT &213s ORGANISM: Olea europaea

<4 OOs, SEQUENCE: 44

Met Gly Ala Gly Gly Arg Lell Ser Wall Pro Ala Thir Ala Glu Glu 1. 5 1O 15

Lys Lys Asn Pro Lell Arg Wall Pro Tyr Luell Pro Pro Phe Thir 2O 25

Wall Gly Asp Ile Lys Thir Ile Pro Pro His Phe Arg Ser 35 4 O 45

Lell Luell Arg Ser Phe Ser Tyr Wall Wall Tyr Asp Lell Phe Luell Wall Phe SO 55 6 O

Lell Phe Ile Ala Thir Ser Phe His Lell Lell Pro Ser Pro 65 70

Phe Ser Luell Gly Trp Ser Wall Trp Ile Lell Glin Gly Cys Wall 85 90 95 US 9,315,837 B2 75 76 - Continued

Thir Gly Wall Trp Wall Ile Ala His Glu Cys Gly His His Ala Phe 1OO 105 11 O

Ser Asp Tyr Glin Trp Wall Asp Asp Thir Wall Gly Lell Ile Luell His Ser 115 12 O 125

Thir Luell Luell Wall Pro Phe Ser Trp Ser His Arg Arg His 13 O 135 14 O

His Ser Asn Thir Gly Ser Lell Glu Arg Asp Glu Wall Phe Wall Pro Lys 145 150 155 160

Pro Ser Lell Ser Trp Phe Thir Lys Lell Asn Asn Pro Pro 1.65 17s

Gly Arg Wall Met Thir Lell Wall Ile Thir Luell Thir Lell Gly Trp Pro Luell 18O 185 19 O

Luell Ala Luell Asn Wall Ser Gly Arg Pro Asp Arg Phe Ala Cys 195

His Tyr Asp Pro His Gly Pro Ile Asn Asp Arg Glu Arg Luell Glin 21 O 215

Ile Ile Ser Asp Wall Wall Ile Ala Thir Ser Ile Luell Tyr 225 23 O 235 24 O

Arg Wall Ala Luell Ala Glin Gly Luell Wall Trp Luell Thir Wall Tyr Gly 245 250 255

Wall Pro Luell Luell Ile Wall Asn Gly Phe Luell Wall Lell Ile Thir Luell 26 O 265 27 O

Glin His Thir His Pro Pro Lell Pro His Asp Ser Ser Glu Trp Asp 27s 28O 285

Trp Lieu Arg Gly Ala Lieu Ala Thr Wall Asp Arg Asp Gly Wall Lieu 29 O 295 3 OO

Asn Asn Wall Phe His Asn Ile Thir Asp Thir His Wall Ala His His Luell 3. OS 310 315

Phe Ser Thir Met Pro His His Ala Met Glu Ala Thir Ala Ile 3.25 330 335

Pro Luell Luell Gly Glu Glin Ser Asp Gly Thir Pro Phe Tyr 34 O 345 35. O

Ala Met Trp Arg Glu Ala Lys Glu Cys Luell Wall Glu Pro Asp 355 360 365

Glu Pro Asn Gly Wall Phe Trp ASn Lys Phe 37 O 375 38O

<210s, SEQ ID NO 45 &211s LENGTH: 382 212. TYPE : PRT &213s ORGANISM: Spinacia oleracea

<4 OOs, SEQUENCE: 45

Met Gly Ala Gly Gly Arg Ser Ile Pro Pro Ser Ala Arg Glu Lys 1. 5 1O 15

Ser Asp Ala Luell Asn Arg Wall Pro Tyr Glu Pro Pro Phe Thir Luell 25 3O

Gly Glin Ile Lys Ala Ile Pro Pro His Phe Lys Arg Ser Wall 35 4 O 45

Lell Arg Ser Phe Ser Wall Wall Asp Phe Thir Ile Ala Phe Luell SO 55 6 O

Lell Wall Ala Thir Asn Ile His Luell Lell Pro Pro Phe 65 70 7s 8O

Asn Luell Ala Trp Pro Wall Gly Phe Wall Glin Gly Wall Luell 85 90 95 US 9,315,837 B2 77 - Continued Thr Gly Val Trp Val Ile Ala His Glu. Cys Gly His His Ala Phe Ser 1OO 105 11 O Asp Tyr Glin Trp Lieu. Asp Asp Thr Val Gly Lieu Val Lieu. His Ser Phe 115 12 O 125 Lieu. Leu Val Pro Tyr Phe Ser Trp Llys Tyr Ser His Arg Arg His His 13 O 135 14 O Ser Asn Thr Gly Ser Met Glu Lys Asp Glu Val Phe Val Pro Glin Arg 145 150 155 160 Lys Glu Asn Met Ser Trp Phe Ser Lys Tyr Lieu Ser Asn Pro Pro Gly 1.65 17O 17s Arg Ile Lieu. Thir Lieu Val Val Thr Lieu. Thir Lieu. Gly Trp Pro Leu Tyr 18O 185 19 O Lieu. Lieu. Phe Asin Val Ser Gly Arg Llys Tyr Glu Arg Phe Ala Cys His 195 2OO 2O5 Tyr Asp Pro Ser Ser Pro Ile Tyr Ser Asp Arg Glu Arg Lieu. Glin Ile 21 O 215 22O Phe Ile Ser Asp Val Gly Ile Ser Ile Val Ala Phe Gly Lieu. Tyr His 225 23 O 235 24 O Lieu Ala Ala Ala Lys Gly Ile Ser Trp Val Lieu. Cys Val Tyr Gly Gly 245 250 255 Pro Leu Lieu Val Val Asn Gly Phe Leu Val Lieu. Ile Thr Phe Leu Gln 26 O 265 27 O His Thr His Pro Ser Leu Pro His Tyr Asp Thr Ser Glu Trp Asp Trp 27s 28O 285 Lieu. Arg Gly Ala Lieu Ala Thr Ala Asp Arg Asp Tyr Gly Ile Lieu. ASn 29 O 295 3 OO Llys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His Lieu. Ile 3. OS 310 315 32O Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala Ile Llys 3.25 330 335 Pro Ile Leu Gly Lys Tyr Tyr Arg Lieu. Asp Ser Thr Pro Val Phe Lys 34 O 345 35. O Ala Met Trp Arg Glu Ala Lys Glu. Cys Met Tyr Val Glu Ala Asp Glu 355 360 365 Asp Asp Glin Asn Lys Gly Val Lieu. Trp Tyr Arg Asn Llys Lieu. 37 O 375 38O

<210s, SEQ ID NO 46 &211s LENGTH: 481 212. TYPE: PRT <213> ORGANISM: Marchantia polymorpha <4 OOs, SEQUENCE: 46 Met Ala Ser Ser Thr Thr Thr Ala Val Lys Glin Ser Ser Gly Gly Lieu. 1. 5 1O 15 Trp Ser Lys Trp Gly Thr Gly Ser Asn Lieu Ser Phe Val Ser Arg Lys 2O 25 3O

Glu Glin Glin Glin Glin Glin Glin Glin Ser Ser Pro Glu Ala Ser Thr Pro 35 4 O 45

Ala Ala Glin Glin Glu Lys Ser Ile Ser Arg Glu Ser Ile Pro Glu Gly SO 55 6 O

Phe Lieu. Thr Val Glu Glu Val Ser Lys His Asp ASn Pro Ser Asp Cys 65 70 7s 8O

Trp Ile Val Ile Asn Asp Llys Val Tyr Asp Val Ser Ala Phe Gly Lys 85 90 95 US 9,315,837 B2 79 80 - Continued

Thir His Pro Gly Gly Pro Wall Ile Phe Thir Glin Ala Gly Arg Asp Ala 105 11 O

Thir Asp Ser Phe Wall Phe His Ser Ala Ala Trp Glin Phe Luell 115 12 O 125

Glin Asp Luell Tyr Ile Gly Asp Luell Asn Ala Glu Pro Wall Ser Glu 13 O 135 14 O

Lell Wall Asp Tyr Arg Asp Luell Thir Ala Phe Met Arg Ser Glin 145 150 155 160

Lell Phe Ser Ser Met Wall Thir Wall Thir Asn 1.65 17O 17s

Phe Ala Ile Luell Ala Ala Ser Luell Ala Wall Ile Ala Trp Ser Glin Thir 18O 185 19 O

Luell Ala Wall Lell Ser Ser Phe Luell Luell Ala Lell Phe Trp Glin 195

Glin Cys Gly Trp Lell Ser His Asp Phe Luell His His Glin Wall Thir Glu 21 O 215 22O

Asn Arg Ser Luell Asn Thir Phe Gly Gly Luell Phe Trp Gly Asn Phe 225 23 O 235 24 O

Ala Glin Gly Tyr Ser Wall Trp Trp Lys Thir His Asn Wall His 245 250 255

His Ala Ala Thir Asn Glu Asp Asp Glin Pro Ile Asp Pro 26 O 265 27 O

Asp Ile Asp Thir Wall Pro Lell Luell Ala Trp Ser Glu Ile Luell Ala 27s 285

Thr Wall Asp Asp Gln Phe Phe Arg Ser Ile Ile Ser Wall Gln His Lieu 29 O 295 3 OO

Lell Phe Phe Pro Lell Lell Phe Luell Ala Arg Phe Ser Trp Luell His Ser 3. OS 310 315

Ser Trp Ala His Ala Ser Asn Phe Glu Met Pro Arg Met Arg Trp 3.25 330 335

Ala Glu Ala Ser Lell Lell Gly His Gly Ala Ser Ile Gly Ala 34 O 345 35. O

Ala Phe Tyr Ile Lell Pro Ile Pro Glin Ala Ile Trp Luell Phe Luell 355 360 365

Ser Glin Luell Phe Gly Ala Luell Luell Ser Ile Wall Phe Wall Ile Ser 37 O 375 38O

His Asn Gly Met Asp Wall Asn Asp Pro Arg Asp Phe Wall Thir Ala 385 390 395 4 OO

Glin Wall Thir Ser Thir Arg Asn Ile Glu Gly ASn Phe Phe Asn Asp Trp 4 OS 415

Phe Thir Gly Gly Lell Asn Arg Glin Ile Glu His His Lell Phe Pro Ser 425 43 O

Lell Pro Arg His Asn Lell Ala Lys Wall Ala Pro His Wall Ala Luell 435 44 O 445

Ala His Gly Lell His Glu Glu Luell Ser Lell Gly Thir Gly 450 45.5 460

Wall Arg Wall Phe Asn Arg Luell Wall Glu Wall Ala Ala Ala Lys 465 470 47s 48O

Wall

<210s, SEQ ID NO 47 &211s LENGTH: 477 212. TYPE : PRT <213> ORGANISM: Phaeodactylum tricornutum US 9,315,837 B2 81 82 - Continued

<4 OOs, SEQUENCE: 47

Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser Thir Ala Ala 1. 15

Arg Lys Ile Ser Trp Glin Glu Wall Lys Thir His Ala Ser Pro Glu Asp 2O 25

Ala Trp Ile Ile His Ser Asn Lys Wall Tyr Asp Wall Ser Asn Trp His 35 4 O 45

Glu His Pro Gly Gly Ala Wall Ile Phe Thir His Ala Gly Asp Asp Met SO 55 6 O

Thir Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser Glin Ser Luell Met 65 70

Phe Ile Gly Glu Luell Luell Pro Glu Thir Thir Gly Lys Glu 85 90 95

Pro Glin Glin Ile Ala Phe Glu Gly Arg Asp Lell Arg Ser Lys 105 11 O

Lell Ile Met Met Gly Met Phe Lys Ser Asn Trp Phe Wall Tyr 115 12 O 125

Cys Luell Ser Asn Met Ala Ile Trp Ala Ala Ala Ala Luell Wall 13 O 135 14 O

Phe Ser Asp Arg Phe Trp Wall His Luell Ala Ser Ala Wall Met Luell 145 150 155 160

Gly Thir Phe Phe Glin Glin Ser Gly Trp Luell Ala His Asp Phe Luell His 1.65 17O 17s

His Glin Wall Phe Thir Arg His Gly Asp Lell Gly Gly Luell Phe 18O 185 19 O

Trp Gly Asn Luell Met Glin Gly Tyr Ser Wall Glin Trp Trp Asn Lys 195 2OO

His Asn Gly His His Ala Wall Pro Asn Luell His Cys Ser Ser Ala Wall 21 O 215 22O

Ala Glin Asp Gly Asp Pro Asp Ile Asp Thir Met Pro Lell Luell Ala Trp 225 23 O 235 24 O

Ser Wall Glin Glin Ala Glin Ser Arg Glu Luell Glin Ala Asp Gly Lys 245 250 255

Asp Ser Gly Luell Wall Phe Met Ile Arg ASn Glin Ser Tyr Phe Tyr 26 O 265 27 O

Phe Pro Ile Luell Lell Lell Ala Arg Luell Ser Trp Lell Asn Glu Ser Phe 28O 285

Cys Ala Phe Gly Lell Gly Ala Ala Ser Glu Asn Ala Ala Luell Glu 29 O 295 3 OO

Lell Ala Gly Lell Glin Pro Luell Luell Glu Ala Ile 3. OS 310 315

Lell Luell His Ala Trp Met Luell Thir Wall Ser Ser Gly Phe Arg 3.25 330

Phe Ser Phe Ala Tyr Thir Ala Phe Tyr Phe Luell Thir Ala Thir Ser 34 O 345 35. O

Gly Phe Luell Lell Ala Ile Wall Phe Gly Luell Gly His Asn Met 355 360 365

Ala Thir Asn Ala Asp Ala Arg Pro Asp Phe Trp Luell Wall 37 O 375

Thir Thir Thir Arg Asn Wall Thir Gly Gly His Gly Phe Pro Glin Phe 385 390 395 4 OO

Wall Asp Trp Phe Cys Gly Gly Luell Glin Tyr Glin Wall Asp His Luell 4 OS 41O US 9,315,837 B2 83 - Continued Phe Pro Ser Lieu Pro Arg His Asn Lieu Ala Lys Thr His Ala Lieu Val 42O 425 43 O Glu Ser Phe Cys Lys Glu Trp Gly Val Glin Tyr His Glu Ala Asp Lieu. 435 44 O 445 Val Asp Gly Thr Met Glu Val Lieu. His His Lieu. Gly Ser Val Ala Gly 450 45.5 460 Glu Phe Val Val Asp Phe Val Arg Asp Gly Pro Ala Met 465 470 47s

<210s, SEQ ID NO 48 &211s LENGTH: 484 212. TYPE: PRT <213> ORGANISM: Thalassiosira pseudonana <4 OOs, SEQUENCE: 48 Met Gly Lys Gly Gly Asp Ala Ala Ala Ala Thr Lys Arg Ser Gly Ala 1. 5 1O 15 Lieu Lys Lieu Ala Glu Lys Pro Glin Llys Tyr Thir Trp Glin Glu Val Lys 2O 25 3O Llys His Ile Thr Pro Asp Asp Ala Trp Val Val His Glin Asn Llys Val 35 4 O 45 Tyr Asp Val Ser Asn Trp Tyr Asp His Pro Gly Gly Ala Val Val Phe SO 55 6 O Thir His Ala Gly Asp Asp Met Thr Asp Ile Phe Ala Ala Phe His Ala 65 70 7s 8O Glin Gly Ser Glin Ala Met Met Lys Llys Phe Tyr Ile Gly Asp Lieu. Ile 85 90 95 Pro Glu Ser Val Glu. His Lys Asp Glin Arg Glin Lieu. Asp Phe Glu Lys 1OO 105 11 O Gly Tyr Arg Asp Lieu. Arg Ala Lys Lieu Val Met Met Gly Met Phe Lys 115 12 O 125 Ser Ser Lys Met Tyr Tyr Ala Tyr Lys Cys Ser Phe Asn Met Cys Met 13 O 135 14 O Trp Lieu Val Ala Val Ala Met Val Tyr Tyr Ser Asp Ser Lieu Ala Met 145 150 155 160 His Ile Gly Ser Ala Lieu Lleu Lieu. Gly Lieu. Phe Trp Glin Glin Cys Gly 1.65 17O 17s Trp Lieu Ala His Asp Phe Lieu. His His Glin Val Phe Lys Glin Arg Llys 18O 185 19 O Tyr Gly Asp Leu Val Gly Ile Phe Trp Gly Asp Leu Met Glin Gly Phe 195 2OO 2O5 Ser Met Gln Trp Trp Lys Asn Llys His Asn Gly His His Ala Val Pro 21 O 215 22O Asn Lieu. His Asn. Ser Ser Lieu. Asp Ser Glin Asp Gly Asp Pro Asp Ile 225 23 O 235 24 O

Asp Thr Met Pro Lieu. Lieu Ala Trp Ser Lieu Lys Glin Ala Glin Ser Phe 245 250 255

Arg Glu Ile Asn Lys Gly Lys Asp Ser Thr Phe Wall Lys Tyr Ala Ile 26 O 265 27 O

Llys Phe Glin Ala Phe Thr Tyr Phe Pro Ile Leu Lleu Lleu Ala Arg Ile 27s 28O 285

Ser Trp Lieu. Asn. Glu Ser Phe Llys Thr Ala Phe Gly Lieu. Gly Ala Ala 29 O 295 3 OO

Ser Glu Asn Ala Lys Lieu. Glu Lieu. Glu Lys Arg Gly Lieu. Glin Tyr Pro 3. OS 310 315 32O US 9,315,837 B2 85 - Continued Lieu. Leu Glu Lys Lieu. Gly Ile Thr Lieu. His Tyr Thr Trp Met Phe Val 3.25 330 335 Lieu. Ser Ser Gly Phe Gly Arg Trp Ser Leu Pro Tyr Ser Ile Met Tyr 34 O 345 35. O Phe Phe Thr Ala Thr Cys Ser Ser Gly Leu Phe Leu Ala Leu Val Phe 355 360 365 Gly Lieu. Gly His Asn Gly Met Ser Val Tyr Asp Ala Thr Thr Arg Pro 37 O 375 38O Asp Phe Trp Gln Leu Glin Val Thir Thr Thr Arg Asn Ile Ile Gly Gly 385 390 395 4 OO His Gly Ile Pro Glin Phe Phe Val Asp Trp Phe Cys Gly Gly Leu Gln 4 OS 41O 415 Tyr Glin Val Asp His His Leu Phe Pro Met Met Pro Arg Asn Asn Ile 42O 425 43 O Ala Lys Cys His Llys Lieu Val Glu Ser Phe Cys Lys Glu Trp Gly Val 435 44 O 445 Llys Tyr His Glu Ala Asp Met Trp Asp Gly Thr Val Glu Val Lieu. Glin 450 45.5 460 His Lieu. Ser Llys Val Ser Asp Asp Phe Lieu Val Glu Met Wall Lys Asp 465 470 47s 48O

Phe Pro Ala Met

<210s, SEQ ID NO 49 &211s LENGTH: 449 212. TYPE: PRT <213> ORGANISM: Mantoniella squamata <4 OOs, SEQUENCE: 49 Met Cys Pro Pro Lys Glu Ser Thr Arg Lys Asn Ala Gly Gly Pro Leu 1. 5 1O 15 Thir Arg Gly Lys Lieu. Ser Ala Asp Lieu Ala Lys Lieu. Glu Pro His Lys 2O 25 3O Lieu Ala Glin Thir Phe Asp Thr Arg Trp Val Arg Val Gly Asp Val Glu 35 4 O 45 Tyr Asp Val Thr Asn Phe Lys His Pro Gly Gly Ser Val Ile Phe Tyr SO 55 6 O

Met Lieu. Ser Asn Thr Gly Ala Asp Ala Thr Glu Ala Phe Asin Glu Phe 65 70 7s 8O His Met Arg Ser Pro Lys Ala Trp Llys Met Lieu Lys Ala Lieu Pro Asn 85 90 95

Arg Pro Ala Glu Thr Pro Arg Ser Glin Asp Pro Asp Gly Pro Met Lieu 1OO 105 11 O

Glu Asp Phe Ala Lys Trp Arg Ala Glin Lieu. Glu Lys Glu Gly Phe Phe 115 12 O 125

Llys Pro Ser Ile Ala His Val Ala Tyr Arg Ile Ala Glu Lieu Ala Ala 13 O 135 14 O

Met Phe Ala Leu Gly Cys Tyr Ile Met Ser Leu Gly Tyr Pro Val Val 145 150 155 160

Ala Ser Ile Val Phe Gly Ala Phe Phe Gly Ala Arg Cys Gly Trp Val 1.65 17O 17s Glin His Glu Gly Gly His Asn. Ser Lieu. Thr Gly Asn. Ile Trp Lieu. Asp 18O 185 19 O

Lys Arg Ile Glin Ala Ala Thr Cys Gly Phe Gly Lieu. Ser Thir Ser Gly 195 2OO 2O5 US 9,315,837 B2 87 88 - Continued

Asp Met Trp Asn Glin Met His Asn His His Ala Thir Pro Glin Lys 21 O 215

Wall Arg His Asp Met Asp Lell Asp Thir Thir Pro Ala Wall Ala Phe Phe 225 23 O 235 24 O

Thir Ala Wall Glu Asp Asn Arg Pro Arg Gly Phe Ser Arg Ala Trp 245 250 255

Ser Arg Ala Glin Ala Trp Thir Phe Wall Pro Wall Thir Ser Gly Luell Luell 26 O 265 27 O

Wall Glin Met Phe Trp Ile Wall Luell His Pro Arg Glin Wall Ala Arg 285

Lys Asn Glu Glu Ala Ser Trp Met Ile Lell Ser His Wall Luell 29 O 295 3 OO

Arg Thir Ala Thir Ile Lys Ala Gly Gly Tyr Ser Trp Pro Wall Ala 3. OS 310 315

Luell Trp Phe Ser Phe Gly Asn Trp Ile Ala Met Luell Phe 3.25 330 335

Ala His Phe Ser Thir Ser His Thir His Luell Glu Wall Wall Pro Ser Asp 34 O 345 35. O

His Ile Ser Trp Wall Asn Tyr Ala Wall Asp His Thir Wall Asp Ile 355 360 365

Asp Pro Ser Gly Wall Asn Trp Luell Met Gly Luell Asn Cys 37 O 375 38O

Glin Wall Ile His His Lell Phe Pro Asp Met Pro Glin Phe Arg Glin Pro 385 390 395 4 OO

Glu Wall Ser Arg Arg Phe Wall Ala Phe Ala Trp Asn Lieu Asn 4 OS 415

Wall Luell Thir Gly Ala Trp Ala Thir Phe Thir Asn 425 43 O

Lell Asp Thir Wall Gly Glin His Tyr Lys His Gly Lys Ala His Ala 435 44 O 445

His

<210s, SEQ ID NO 50 &211s LENGTH: 456 212. TYPE : PRT <213> ORGANISM: Ostreococcus tauri

<4 OOs, SEQUENCE: SO

Met Cys Val Glu Thir Glu Asn Asn Asp Gly Ile Pro Thir Wall Glu Ile 1. 5 15

Ala Phe Asp Gly Glu Arg Glu Arg Ala Glu Ala Asn Wall Lys Luell Ser 25

Ala Glu Lys Met Glu Pro Ala Ala Luell Ala Thir Phe Ala Arg Arg 35 4 O 45

Wall Wall Ile Glu Gly Wall Glu Asp Wall Thir Asp Phe His SO 55 6 O

Pro Gly Gly Thir Wall Ile Phe Ala Luell Ser Asn Thir Gly Ala Asp 65 70

Ala Thir Glu Ala Phe Glu Phe His His Arg Ser Arg Ala Arg 85 90 95

Ala Luell Ala Ala Lell Pro Ser Arg Pro Ala Thir Ala Wall 105 11 O

Asp Asp Ala Glu Met Lell Glin Asp Phe Ala Lys Trp Arg Glu Luell 115 12 O 125 US 9,315,837 B2 89 - Continued Glu Arg Asp Gly Phe Phe Llys Pro Ser Pro Ala His Val Ala Tyr Arg 13 O 135 14 O Phe Ala Glu Lieu Ala Ala Met Tyr Ala Leu Gly Thr Tyr Lieu Met Tyr 145 150 155 160 Ala Arg Tyr Val Val Ser Ser Val Lieu Val Tyr Ala Cys Phe Phe Gly 1.65 17O 17s Ala Arg Cys Gly Trp Val Gln His Glu Gly Gly His Ser Ser Lieu. Thr 18O 185 19 O Gly Asn. Ile Trp Trp Asp Lys Arg Ile Glin Ala Phe Thr Ala Gly Phe 195 2OO 2O5 Gly Lieu Ala Gly Ser Gly Asp Met Trp Asn. Ser Met His Asn Llys His 21 O 215 22O His Ala Thr Pro Glin Llys Val Arg His Asp Met Asp Lieu. Asp Thir Thr 225 23 O 235 24 O Pro Ala Val Ala Phe Phe Asn. Thir Ala Val Glu Asp Asn Arg Pro Arg 245 250 255 Gly Phe Ser Lys Tyr Trp Lieu. Arg Lieu. Glin Ala Trp Thr Phe Ile Pro 26 O 265 27 O Val Thr Ser Gly Lieu Val Lieu. Leu Phe Trp Met Phe Phe Lieu. His Pro 27s 28O 285 Ser Lys Ala Lieu Lys Gly Gly Lys Tyr Glu Glu Lieu Val Trp Met Lieu. 29 O 295 3 OO Ala Ala His Val Ile Arg Thir Trp Thir Ile Lys Ala Val Thr Gly Phe 3. OS 310 315 32O Thr Ala Met Glin Ser Tyr Gly Lieu Phe Lieu Ala Thr Ser Trp Val Ser 3.25 330 335 Gly Cys Tyr Lieu Phe Ala His Phe Ser Thr Ser His Thr His Lieu. Asp 34 O 345 35. O Val Val Pro Ala Asp Glu. His Lieu. Ser Trp Val Arg Tyr Ala Val Asp 355 360 365 His Thr Ile Asp Ile Asp Pro Ser Glin Gly Trp Val Asn Trp Leu Met 37 O 375 38O Gly Tyr Lieu. Asn Cys Glin Val Ile His His Leu Phe Pro Ser Met Pro 385 390 395 4 OO Glin Phe Arg Glin Pro Glu Val Ser Arg Arg Phe Val Ala Phe Ala Lys 4 OS 41O 415 Llys Trp Asn Lieu. Asn Tyr Llys Val Met Thr Tyr Ala Gly Ala Trp Llys 42O 425 43 O Ala Thr Lieu. Gly Asn Lieu. Asp Asn Val Gly Llys His Tyr Tyr Val His 435 44 O 445 Gly Glin His Ser Gly Lys Thr Ala 450 45.5

<210s, SEQ ID NO 51 &211s LENGTH: 482 212. TYPE: PRT <213> ORGANISM: Mantoniella squamata

<4 OOs, SEQUENCE: 51 Met Pro Pro Arg Glu Thir Thr Thr Pro Ser Val Asp His Pro Val Met 1. 5 1O 15

Asp Arg Ile Thir Ser Lieu. Thr Gly Gly Ala Gly Ala Gly Val Pro Arg 2O 25 3O Llys Tyr Thir Thir Ala Asp Val Glu Lys His Ser Thr Pro Asp Asp Cys 35 4 O 45 US 9,315,837 B2 91 92 - Continued

Trp Luell Ile Wall His Gly Lys Wall Asp Wall Thir Ser Phe Wall Pro SO 55 6 O

Arg His Pro Gly Gly Asn Met Ile Trp Wall Lys Ala Gly Gly Asp Cys 65 70 7s 8O

Thir Glin Luell Phe Asp Ser His Pro Ile Thir Glin Ala Wall Luell 85 90 95

Asp Tyr Ile Gly Glu Wall Glin Arg Wall Ser Gly Asp Glu 105 11 O

Ile Ile Glu Tyr Asn Asp Asp Met Gly Lys Phe Met 115 12 O 125

Asp Cys Wall Ala Wall Glu Phe Asp Thir Glin Asp 13 O 135 14 O

Pro Arg Wall His Wall Glu Met Wall Thir Phe Wall Ile Luell Ala 145 150 155 160

Gly Wall Ala Wall Cys His Ser Phe Phe Lell Thir Ser Ser Phe 1.65 17O 17s

Lell Wall Ser Ala Wall Phe Ala Ala Luell His Gly Met Trp Lys Ala Glu 18O 185 19 O

Wall Gly Wall Ser Ile Glin His Asp Ala Asn His Gly Ala Gly 195 2OO

Ser Arg Gly Phe Lell His Ala Met Glin Luell Thir Lell Asp Wall Wall Gly 21 O 215

Ala Ser Ser Phe Met Trp Arg Glin Glin His Wall Wall Gly His His Ala 225 23 O 235 24 O

Thir Asn Wall Glu Gly Wall Asp Pro Asp Ile Arg Ala Pro Glu 245 250 255

Asp Ile Arg Arg Wall Asn Glu His Glin Pro His Glu Ser His 26 O 265 27 O

Pro Luell Glin His Wall Lell Phe Phe Ala Gly Lell Luell Ser Phe 27s 28O 285

Ser Phe Ala Asp Asp Phe Asn Ala Trp Ala Ser Gly Arg Ile 29 O 295 3 OO

Gly Trp Wall Wall Ala Phe Thir Arg Gly Glu Ala Wall Ser Phe 3. OS 310 315

Trp Gly Ser Wall Lell Trp Ala Phe Tyr Lell Luell Pro Ala 3.25 330 335

Thir Ser Pro His Ser Gly Luell Arg Ile Wall Ala Lell Wall Thir Ile 34 O 345 35. O

Thir Glu Wall Ile Thir Gly Trp Luell Luell Ala Phe Met Phe Glin Wall Ala 355 360 365

His Wall Wall Gly Asp Wall Arg Phe Phe Luell Ser Glu Glu Gly 37 O 375

Lell Asn Luell Gly Trp Gly Glu Ser Glin Luell Tyr Ser Ser Ala Asp Phe 385 390 395 4 OO

Ala His Gly Ser Lys Phe Trp Met His Phe Ser Gly Gly Luell Asn 4 OS 415

Glin Wall Ala His His Lell Phe Pro Gly Wall Cys His His Pro 425 43 O

Ala Ile Ala Pro Ile Ile Met Lys Wall Ala Lys Glu Tyr Gly Luell Glu 435 44 O 445

Ala Wall Tyr Pro Thir Phe Trp Ser Ala Luell Ser Ala His Phe Thir 450 45.5 460 US 9,315,837 B2 93 94 - Continued His Leu Lys Asn Val Gly Gln Lys Thr Tyr Val Pro Ser Leu Glin Thr 465 470 47s 48O Ile Gly

<210s, SEQ ID NO 52 &211s LENGTH: 491 212. TYPE: PRT <213> ORGANISM: Ostreococcus tauri

<4 OOs, SEQUENCE: 52 Met Gly. Thir Thr Ala Arg Asp Ala Gly Ala Val Thir Thr Arg Ala Arg 1. 5 1O 15 Arg Arg Gly Thr Gly Ala Thir Ser Glu Ala Ser Arg Val Val His Ala 2O 25 3O Val Asp Ala Asp Ala Arg Thr Tyr Thr Ala Ala Glu Val Ala Thr His 35 4 O 45 Ala Arg Ala Asp Asp Cys Trp Val Ile Val Arg Gly Gly Val Tyr Asp SO 55 6 O Val Thr Ala Phe Val Pro Arg His Pro Gly Gly Asn Met Ile Tyr Val 65 70 7s 8O Lys Ala Gly Gly Glu. Cys Thr Ala Lieu. Phe Asp Ser Tyr His Pro Glu 85 90 95 Lys Ala Arg Gly Val Lieu. Glu Lys Tyr Arg Ile Gly Asp Lieu. Thir Arg 1OO 105 11 O Glu Glu Gly Ser Ala Ala Asp Gly Asp Ile Val Glu Tyr Ala Lys Asp 115 12 O 125 Asp Lieu Lys Asp Gly Ala Phe Phe Ala Asp Cys Lys Ala Gly Ala Ala 13 O 135 14 O Llys Tyr Phe Lys Glu Asn Llys Lieu. Asp Pro Arg Val His Trp Glu Met 145 150 155 160 Tyr Ala Lys Thir Ala Ala Ile Lieu Val Gly Ile Val Val Gly His Tyr 1.65 17O 17s Tyr Ser Phe Phe Ala Pro Gly Val Ser Phe Gly Ala Ala Leu Ala Phe 18O 185 19 O

Ala Ala Lieu. His Gly. Thir Cys Lys Ala Glu Val Gly Val Ser Ile Glin 195 2OO 2O5 His Asp Ala Asn His Gly Ala Tyr Gly Asn. Ser Arg Thir Trp Lieu. His 21 O 215 22O

Ala Met Gln Lieu. Thir Lieu. Asp Val Val Gly Ala Ser Ser Phe Met Trp 225 23 O 235 24 O

Lys Glin Gln His Val Ala Gly His His Ala Tyr Thr Asn Val Glu Gly 245 250 255

Ile Asp Pro Asp Ile Arg Cys Ser Glu Lys Asp Ile Arg Arg Val Asn 26 O 265 27 O

Glu. His Gln Pro His Glu Pro Tyr His Val Phe Gln His Val Tyr Lieu. 27s 28O 285

Ala Phe Met Tyr Gly Lieu Lleu Ser Lieu Lys Ser Cys Phe Val Asp Asp 29 O 295 3 OO

Phe Asn Ala Tyr Phe Ser Gly Arg Ile Gly Trp Val Llys Val Met Lys 3. OS 310 315 32O

Phe Thr Arg Gly Glu Ala Ile Ala Phe Trp Gly Thr Llys Lieu. Lieu. Trp 3.25 330 335

Ala Ala Tyr Tyr Lieu Ala Lieu Pro Lieu Lys Met Ser His Arg Pro Lieu 34 O 345 35. O US 9,315,837 B2 95 96 - Continued

Gly Glu Luell Luell Ala Lell Trp Ala Wall Thir Glu Phe Wall Thir Gly Trp 355 360 365

Lell Luell Ala Phe Met Phe Glin Wall Ala His Wall Wall Gly Glu Wall His 37 O 375

Phe Phe Thir Luell Asp Ala Asn Arg Wall ASn Lell Gly Trp Gly Glu 385 390 395 4 OO

Ala Glin Luell Met Ser Ser Ala Asp Phe Ala His Gly Ser Phe Trp 4 OS 415

Thir His Phe Ser Gly Gly Lell Asn Tyr Glin Wall Wall His His Luell Phe 42O 425 43 O

Pro Gly Wall His Wall His Tyr Pro Ala Luell Ala Pro Ile Ile 435 44 O 445

Ala Ala Ala Glu His Gly Luell His Tyr Glin Ile Pro Thir Phe 450 45.5 460

Trp Ser Ala Luell Arg Ala His Phe Arg His Luell Ala Asn Wall Gly Arg 465 470 48O

Ala Ala Wall Pro Ser Lell Glin Thir Wall Gly 485 490

<210s, SEQ ID NO 53 &211s LENGTH: 484 212. TYPE : PRT &213s ORGANISM: Marchantia polymorpha

<4 OOs, SEQUENCE: 53

Met Pro Pro His Ala Pro Asp Ser Thir Gly Luell Gly Pro Glu Wall Phe 1. 5 1O 15

Arg Luell Pro Asp Asp Ala Ile Pro Ala Glin Asp Arg Arg Ser Thir Glin 25

Tyr Ser Lell Ser Asp Wall Ser His Asn Thir Pro Asn Asp 35 4 O 45

Trp Luell Wall Ile Trp Gly Wall Tyr Asp Wall Thir Ser Trp Wall SO 55 6 O

Lys Wall His Pro Gly Gly Ser Luell Ile Phe Wall Ala Gly Glin Asp 65 70

Ser Thir Glin Luell Phe Asp Ser His Pro Luell Tyr Wall Arg Lys Luell 85 90 95

Lell Ala Glin Phe Ile Gly Glu Luell Glin Thir Ser Ala Gly Asp Glu 105 11 O

Phe Lys Ser Ser Thir Lell Glu Tyr Ala Gly Glu Glu His Glu Wall 115 12 O 125

Phe Tyr His Thir Lell Glin Arg Wall Glu Thir Tyr Phe Arg Glin 13 O 135 14 O

Lys Ile Asn Pro Arg Tyr His Pro Glin Met Luell Wall Ser Ala Wall 145 150 155 160

Ile Ile Gly Thir Lell Lell Lell Tyr Phe Gly Phe Phe Trp Ser 1.65 17O 17s

Glin Asn Wall Luell Lell Ser Met Phe Luell Ala Ser Ile Met Gly Phe 18O 185 19 O

Thir Ala Glu Wall Gly Met Ser Ile Met His Asp Gly Asn His Gly Ser 195 2OO

Thir Glin Ser Thir Lell Lell Gly Tyr Wall Met Gly Ala Thir Luell Asp 21 O 215 22O

Lell Wall Gly Ala Ser Ser Phe Met Trp Arg Glin Glin His Wall Ala Gly 225 23 O 235 24 O US 9,315,837 B2 97 98 - Continued

His His Ser Phe Thir Asn Ile Asp His Tyr Asp Pro Asp Ile Arg Wall 245 250 255

Asp Pro Asp Lell Arg Arg Wall Thir Ser Glin Glin Pro Arg Arg Trp 26 O 265 27 O

Phe His Glu Tyr Glin His Ile Tyr Luell Gly Wall Lell Tyr Gly Wall Luell 27s 28O 285

Ala Luell Llys Ser Wall Lell Ile Asp Asp Phe Ser Ala Phe Phe Ser Gly 29 O 295 3 OO

Ala Ile Gly Pro Wall Lys Ile Ala Glin Met Thir Pro Lell Glu Met Gly 3. OS 310 315

Wall Phe Trp Gly Gly Wall Wall Ala Luell Tyr Met Phe Luell Luell 3.25 330 335

Pro Met Met Tyr Gly Glin Tyr Asn Ile Luell Thir Phe Ile Gly Luell Tyr 34 O 345 35. O

Ile Luell Ser Glin Lell Wall Ala Gly Trp Thir Luell Ala Lell Phe Phe Glin 355 360 365

Wall Ala His Wall Wall Asp Asp Ala Wall Phe Pro Wall Ala Glu Thir Asp 37 O 375

Gly Gly Lys Ala Ile Pro Ser Gly Trp Ala Glu Met Glin Wall Arg 385 390 395 4 OO

Thir Thir Thir Asn Phe Ser Ser Arg Ser Met Phe Trp Thir His Ile Ser 4 OS 415

Gly Gly Luell Asn His Glin Ile Glu His His Luell Phe Pro Gly Wall Cys 42O 425 43 O

His Wall His Tyr Pro Ser Ile Pro Ile Wall Ala Thr Asp 435 44 O 445

Glu Phe Asn. Wall Pro Thir Ser Pro Thir Phe Trp Ala Ala Luell 450 45.5 460

Arg Ala His Phe Glin His Lell Asn Wall Gly Lell Glin Asp Gly Luell 465 470 47s 48O

Arg Luell Asp Gly

<210s, SEQ ID NO 54 &211s LENGTH: 467 212. TYPE: PRT <213> ORGANISM: Dictyostelium discoideum

<4 OOs, SEQUENCE: 54

Met Met Glu. Thir Asn Asn Glu Asn Lys Glu Lys Lell Luell Tyr Thir 1. 5 1O 15

Trp Asp Glu Wall Ser His Asn Glin ASn Asp Lell Trp Ile Ile 2O 25 3O

Wall Asp Gly Lys Wall Asn Ile Thir Trp Wall Pro Luell His Pro 35 4 O 45

Gly Gly Glu Asp Ile Lell Lell Luell Ser Ala Gly Arg Asp Ala Thir Asn SO 55 6 O

Lell Phe Glu Ser Tyr His Pro Met Thir Asp Lys His Ser Luell Ile 65 70

Glin Tyr Glu Ile Gly Tyr Ile Ser Ser Tyr Glu His Pro Lys Tyr 85 90 95

Wall Glu Llys Ser Glu Phe Ser Thir Luell Lys Glin Arg Wall Arg 105 11 O

His Phe Gn. Thir Ser Ser Glin Asp Pro Wall Ser Wall Gly Wall Phe 115 12 O 125 US 9,315,837 B2 99 100 - Continued

Thir Arg Met Wall Lell Ile Tyr Luell Phe Luell Phe Wall Thir Tyr Tyr Luell 13 O 135 14 O

Ser Glin Phe Ser Thir Asp Arg Phe Trp Luell ASn Cys Ile Phe Ala Wall 145 150 155 160

Lell Tyr Gly Wall Ala Asn Ser Luell Phe Gly Luell His Thir Met His Asp 1.65 17O 17s

Ala His Thir Ala Ile Thir His Asn Pro Met Thir Trp Lys Ile Luell 18O 185 19 O

Gly Ala Thir Phe Asp Lell Phe Ala Gly Ala Ser Phe Tyr Ala Trp 195

His Glin His Wall Ile Gly His His Luell Tyr Thir Asn Wall Arg Asn Ala 21 O 215 22O

Asp Pro Asp Luell Gly Glin Gly Glu Ile Asp Phe Arg Wall Wall Thir Pro 225 23 O 235 24 O

Glin Ala Arg Ser Trp His Tyr Glin His Ile Ala Pro 245 250 255

Ile Luell Gly Wall Ala Luell Lys Arg Ile Glin Asp His Glu 26 O 265 27 O

Ile Phe Thir Lys Ser Asn Gly Ala Ile Arg Ser Pro Ile Ser 27s 285

Thir Ile Asp Thir Ala Ile Phe Ile Luell Gly Lys Lell Wall Phe Ile Ile 29 O 295 3 OO

Ser Arg Phe Ile Lell Pro Lell Ile Asn His Ser Phe Ser His Luell 3. OS 310 315

Ile Phe Phe Lieu Ile Ser Glu Lieu Wall Lieu. Gly Trp Lieu Ala 3.25 330 335

Ile Ser Phe Glin Wall Ser His Wall Wall Glu Asp Lell Glin Phe Met Ala 34 O 345 35. O

Thir Pro Glu Ile Phe Asp Gly Ala Asp His Pro Lell Pro Thir Thir Phe 355 360 365

Asn Glin Asp Trp Ala Ile Lell Glin Wall Thir Thir Glin Asp Ala 37 O 375

Glin Asp Ser Wall Lell Ser Thir Phe Phe Ser Gly Gly Lell Asn Luell Glin 385 390 395 4 OO

Wall Ile His His Cys Phe Pro Thir Ile Ala Glin Asp Pro Glin 4 OS 41O 415

Ile Wall Pro Ile Lell Glu Wall Cys Glu Tyr Asn Wall Thir Tyr 425 43 O

His Lys Pro Thir Phe Thir Glu Ala Ile Lys Ser His Ile Asn Tyr 435 44 O 445

Lell Tyr Met Gly Asn Asp Pro Asp Wall Arg Pro Wall Asn 450 45.5 460

Lys Asn Asp 465

<210s, SEQ ID NO 55 &211s LENGTH: 446 212. TYPE : PRT <213> ORGANISM: Mortierella alpina

<4 OOs, SEQUENCE: 55 Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Glu Glu Lieu Ala Ala 1. 5 15 His Asn. Thir Lys Asp Asp Lieu. Lieu. Lieu Ala Ile Arg Gly Arg Val Tyr 25 US 9,315,837 B2 101 102 - Continued

Asp Wall Thir Phe Lell Ser Arg His Pro Gly Gly Val Asp Thr Luell 35 4 O 45

Lell Luell Gly Ala Gly Arg Asp Wall Thir Pro Wall Phe Glu Met Tyr His SO 55 6 O

Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Wall Gly Thir 65 70

Lell Wall Ser Asn Glu Lell Pro Ile Phe Pro Glu Pro Thir Wall Phe His 85 90 95

Thir Ile Lys Thir Arg Wall Glu Gly Tyr Phe Thir Asp Arg Asn Ile 105 11 O

Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly Arg Ala Luell Ile Phe 115 12 O 125

Gly Ser Luell Ile Ala Ser Tyr Ala Glin Luell Phe Wall Pro Phe Wall 13 O 135 14 O

Wall Glu Arg Thir Trp Lell Glin Wall Wall Phe Ala Ile Ile Met Gly Phe 145 150 155 160

Ala Ala Glin Wall Gly Lell Asn Pro Luell His Asp Ala Ser His Phe 1.65 17O 17s

Ser Wall Thir His Asn Pro Thir Wall Trp Lys Ile Lell Gly Ala Thir His 18O 185 19 O

Asp Phe Phe Asn Gly Ala Ser Tyr Luell Wall Trp Met Tyr Glin His Met 195

Lell Gly His His Pro Thir Asn Ile Ala Gly Ala Asp Pro Asp Wall 21 O 215

Ser Thir Ser Glu Pro Asp Wall Arg Arg Ile Lys Pro Asn Glin Trp 225 23 O 235 24 O

Phe Wall Asn His Ile Asn Glin His Met Phe Wall Pro Phe Luell Tyr Gly 245 250 255

Lell Luell Ala Phe Wall Arg Ile Glin Asp Ile Asn Ile Luell Phe 26 O 265 27 O

Wall Thir Asn Asp Ala Ile Arg Wall Asn Pro Ile Ser Thir Trp His 27s 285

Thir Wall Met Phe Trp Gly Gly Ala Phe Phe Wall Trp Arg Luell 29 O 295 3 OO

Ile Wall Pro Luell Glin Tyr Lell Pro Luell Gly Lys Wall Lell Luell Luell Phe 3. OS 310 315

Thir Wall Ala Asp Met Wall Ser Ser Trp Luell Ala Lell Thir Phe Glin 3.25 330 335

Ala Asn His Wall Wall Glu Glu Wall Glin Trp Pro Lell Pro Asp Glu Asn 34 O 345 35. O

Gly Ile Ile Glin Asp Trp Ala Ala Met Glin Wall Glu Thir Thir Glin 355 360 365

Asp Tyr Ala His Asp Ser His Luell Trp Thir Ser Ile Thir Gly Ser Luell 37 O 375

Asn Glin Ala Wall His His Luell Phe Pro ASn Wall Ser Glin His His 385 390 395 4 OO

Pro Asp Ile Lell Ala Ile Ile Asn Thir Ser Glu Tyr Lys 4 OS 415

Wall Pro Luell Wall Asp Thir Phe Trp Glin Ala Phe Ala Ser His 425 43 O

Lell Glu His Luell Arg Wall Lell Gly Luell Arg Pro Glu Glu 435 44 O 445 US 9,315,837 B2 103 104 - Continued

<210s, SEQ ID NO 56 &211s LENGTH: 469 212. TYPE: PRT <213> ORGANISM: Phaeodactylum tricornutum <4 OOs, SEQUENCE: 56 Met Ala Pro Asp Ala Asp Llys Lieu. Arg Glin Arg Glin Thir Thr Ala Val 1. 5 1O 15 Ala Lys His Asn Ala Ala Thir Ile Ser Thr Glin Glu Arg Lieu. Cys Ser 2O 25 3O Lieu. Ser Ser Lieu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr 35 4 O 45 Asp Leu Glin Ser Phe Asp His Pro Gly Gly Glu Thir Ile Llys Met Phe SO 55 6 O Gly Gly Asn Asp Val Thr Val Glin Tyr Lys Met Ile His Pro Tyr His 65 70 7s 8O Thr Glu Lys His Lieu. Glu Lys Met Lys Arg Val Gly Llys Val Thr Asp 85 90 95 Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Llys 1OO 105 11 O Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly. Thir Lieu 115 12 O 125 Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Lieu. 13 O 135 14 O Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Lieu. Leu Ala Val Ala 145 150 155 160 Tyr Gly Ile Ser Glin Ala Met Ile Gly Met Asn Val Gln His Asp Ala 1.65 17O 17s Asn His Gly Ala Thir Ser Lys Arg Pro Trp Val Asn Asp Met Lieu. Gly 18O 185 19 O Lieu. Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Lieu. Trp Glin Glu Glin 195 2OO 2O5 His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp 21 O 215 22O

Ser Phe Gly Ala Glu Pro Met Lieu. Lieu. Phe Asn Asp Tyr Pro Lieu. Asp 225 23 O 235 24 O

His Pro Ala Arg Thr Trp Lieu. His Arg Phe Glin Ala Phe Phe Tyr Met 245 250 255

Pro Val Lieu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Glin Ile 26 O 265 27 O

Lieu. Asp Lieu. Glin Glin Arg Gly Ala Lieu. Ser Val Gly Ile Arg Lieu. Asp 27s 28O 285 Asn Ala Phe Ile His Ser Arg Arg Llys Tyr Ala Val Phe Trp Arg Ala 29 O 295 3 OO

Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly 3. OS 310 315 32O

Lieu. Glu Trp Ser Trp Arg Val Phe Gly Asin Ile Met Leu Met Gly Val 3.25 330 335

Ala Glu Ser Lieu Ala Lieu Ala Wall Lieu. Phe Ser Lieu. Ser His Asn. Phe 34 O 345 35. O

Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Lieu Lys Llys Thr Gly Glu 355 360 365

Pro Val Asp Trp Phe Lys Thr Glin Val Glu Thir Ser Cys Thr Tyr Gly 37 O 375 38O US 9,315,837 B2 105 106 - Continued

Gly Phe Luell Ser Gly Cys Phe Thir Gly Gly Luell Asn Phe Glin Wall Glu 385 390 395 4 OO

His His Luell Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala 4 OS 41O 415

Pro Wall Arg Glu Ile Ala Lys His Gly Wall His Tyr Ala Tyr 425 43 O

Pro Trp Ile His Glin Asn Phe Luell Ser Thir Wall Arg Tyr Met His 435 44 O 445

Ala Ala Gly Thir Gly Ala Asn Trp Arg Glin Met Ala Arg Glu Asn Pro 450 45.5 460

Lell Thir Gly Arg Ala 465

SEO ID NO 57 LENGTH: 292 TYPE : PRT ORGANISM: Ostreococcus tauri

< 4 OOs SEQUENCE:

Met Ser Gly Lieu. Arg Ala Pro Asn Phe Luell His Arg Phe Trp Thir 1. 5 15

Trp Asp Tyr Ala Ile Ser Wall Wall Phe Thir Ala Asp Ser Phe 25

Glin Trp Asp Ile Gly Pro Wall Ser Ser Ser Thir Ala His Luell Pro Ala 35 4 O 45

Ile Glu Ser Pro Thir Pro Lieu Wall Thir Ser Lieu. Lieu Phe Lieu Wall SO 55 6 O

Thir Wall Phe Luell Trp Tyr Gly Arg Luell Thir Arg Ser Ser Asp Lys 65 70

Ile Arg Glu Pro Thir Trp Lell Arg Arg Phe Ile Ile His Asn Ala 85 90 95

Phe Luell Ile Wall Lell Ser Lell Met Luell Gly Wall Ala Glin 105 11 O

Ala Glin Asn Gly Tyr Thir Luell Trp Gly ASn Glu Phe Ala Thir 115 12 O 125

Glu Thir Glin Luell Ala Lell Tyr Ile Ile Phe Tyr Wall Ser Ile 13 O 135 14 O

Tyr Glu Phe Wall Asp Thir Ile Met Luell Luell Asn Asn Luell Arg 145 150 155 160

Glin Wall Ser Phe Lell His Ile His His Ser Thir Ile Ser Phe Ile 1.65 17s

Trp Trp Ile Ile Ala Arg Arg Ala Pro Gly Gly Asp Ala Tyr Phe Ser 18O 185 19 O

Ala Ala Luell Asn Ser Trp Wall His Wall Met Thir Luell 195

Lell Ser Thir Luell Ile Gly Lys Glu Asp Pro Lys Arg Ser Asn Tyr Luell 21 O 215 22O

Trp Trp Gly Arg His Lell Thir Glin Met Glin Met Lell Glin Phe Phe Phe 225 23 O 235 24 O

Asn Wall Luell Glin Ala Lell Ala Ser Phe Ser Thir Pro 245 250 255

Phe Luell Ser Lys Ile Lell Lell Wall Tyr Met Met Ser Lell Luell Gly Luell 26 O 265 27 O US 9,315,837 B2 107 108 - Continued Phe Gly His Phe Tyr Tyr Ser Lys His Ile Ala Ala Ala Lys Lieu. Glin 27s 285 Llys Lys Glin Glin 29 O

<210s, SEQ ID NO 58 &211s LENGTH: 290 212. TYPE : PRT &213s ORGANISM: Marchantia polymorpha

<4 OOs, SEQUENCE: 58

Met Glu Ala Tyr Glu Met Wall Asp Ser Phe Wall Ser Thir Wall Phe 1. 5 15

Glu Thir Luell Glin Arg Lell Arg Gly Gly Wall Wall Lell Thir Glu Ser Ala 2O 25

Ile Thir Lys Gly Lell Pro Wall Asp Ser Pro Thir Pro Ile Wall Luell 35 4 O 45

Gly Luell Ser Ser Tyr Lell Thir Phe Wall Phe Luell Gly Lell Ile Wall Ile SO 55 6 O

Lys Ser Luell Asp Lell Lys Pro Arg Ser Glu Pro Ala Ile Luell Asn 65 70

Lell Phe Wall Ile Phe His Asn Phe Wall Cys Phe Ala Lell Ser Luell Tyr 85 90 95

Met Wall Gly Ile Wall Arg Glin Ala Ile Luell Asn Arg Tyr Ser Luell 105 11 O

Trp Gly Asn Ala Tyr Asn Pro Lys Glu Wall Glin Met Gly His Lieu Lieu 115 12 O 125

Ile Phe Met Ser Lys Ile Glu Phe Met Asp Thir Wall Ile 13 O 135 14 O

Met Ile Luell Asn Thir Arg Glin Ile Thir Wall Lell His Wall Tyr 145 150 155 160

His His Ala Ser Ile Ser Phe Ile Trp Trp Ile Ile Ala His Ala 1.65 17O 17s

Pro Gly Gly Glu Ala Phe Ser Ala Ala Luell Asn Ser Gly Wall His 18O 185 19 O

Wall Luell Met Lell Luell Luell Ala Ala Thir Lell Gly Asn 195

Glu Lys Ala Arg Arg Tyr Luell Trp Trp Gly Lys Luell Thir Glin 21 O 215 22O

Lell Glin Met Phe Glin Phe Wall Luell Asn Met Ile Glin Ala Asp 225 23 O 235 24 O

Ile Asn Asn Ser Pro Pro Glin Phe Luell Ile Glin Ile Luell Phe 245 250 255

Met Ile Ser Lell Lell Ala Luell Phe Gly Asn Phe Tyr Wall His 26 O 265 27 O

Wall Ser Ala Pro Ala Lys Pro Ala Lys Ile Lys Ser Lys 27s 28O 285

Ala Glu 29 O

<210s, SEQ ID NO 59 &211s LENGTH: 290 212. TYPE : PRT <213> ORGANISM: Physcomitrella patens US 9,315,837 B2 109 110 - Continued

<4 OO > SEQUENCE: 59 Met Glu Val Val Glu Arg Phe Tyr Gly Glu Lieu. Asp Gly Llys Val Ser 1. 5 1O 15 Glin Gly Val Asn Ala Lieu. Lieu. Gly Ser Phe Gly Val Glu Lieu. Thir Asp 2O 25 3O Thr Pro Thr Thr Lys Gly Lieu Pro Leu Val Asp Ser Pro Thr Pro Ile 35 4 O 45 Val Lieu. Gly Val Ser Val Tyr Lieu. Thir Ile Val Ile Gly Gly Lieu. Lieu SO 55 6 O Trp Ile Lys Ala Arg Asp Lieu Lys Pro Arg Ala Ser Glu Pro Phe Lieu. 65 70 7s 8O Lieu. Glin Ala Lieu Val Lieu Val His Asn Lieu. Phe Cys Phe Ala Lieu. Ser 85 90 95 Lieu. Tyr Met Cys Val Gly Ile Ala Tyr Glin Ala Ile Thr Trp Arg Tyr 1OO 105 11 O Ser Lieu. Trp Gly Asn Ala Tyr Asn Pro Llys His Lys Glu Met Ala Ile 115 12 O 125 Lieu Val Tyr Lieu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 13 O 135 14 O Val Ile Met Ile Leu Lys Arg Ser Thr Arg Glin Ile Ser Phe Lieu. His 145 150 155 160 Val Tyr His His Ser Ser Ile Ser Lieu. Ile Trp Trp Ala Ile Ala His 1.65 17O 17s His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Lieu. Asn. Ser Gly 18O 185 19 O Val His Val Lieu Met Tyr Ala Tyr Tyr Phe Lieu Ala Ala Cys Lieu. Arg 195 2OO 2O5 Ser Ser Pro Llys Lieu Lys Asn Llys Tyr Lieu. Phe Trp Gly Arg Tyr Lieu. 21 O 215 22O Thr Glin Phe Gln Met Phe Glin Phe Met Lieu. Asn Lieu Val Glin Ala Tyr 225 23 O 235 24 O Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Lieu. Ile Lys Ile 245 250 255

Lieu. Phe Tyr Tyr Met Ile Ser Lieu. Leu Phe Leu Phe Gly Asin Phe Tyr 26 O 265 27 O

Val Glin Llys Tyr Ile Llys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys 27s 28O 285

Thir Glu 29 O

<210s, SEQ ID NO 60 &211s LENGTH: 348 212. TYPE: PRT <213> ORGANISM: Marchantia polymorpha

<4 OOs, SEQUENCE: 60 Met Ala Thir Lys Ser Gly Ser Gly Lieu. Lieu. Glu Trp Ile Ala Val Ala 1. 5 1O 15

Ala Lys Met Lys Glin Ala Arg Ser Ser Pro Glu Gly Glu Ile Val Gly 2O 25 3O

Gly Asn Arg Met Gly Ser Gly Asn Gly Ala Glu Trp Thr Thr Ser Lieu. 35 4 O 45

Ile His Ala Phe Lieu. Asn Ala Thir Asn Gly Lys Ser Gly Gly Ala Ser SO 55 6 O US 9,315,837 B2 111 112 - Continued

Lys Wall Arg Pro Lell Glu Glu Arg Ile Gly Glu Ala Val Phe Arg Val 65 70

Lell Glu Asp Wall Wall Gly Wall Asp Ile Arg Pro Asn Pro Wall Thir 85 90 95

Asp Luell Pro Met Wall Glu Ser Pro Wall Pro Wall Lell Ala Ile 105 11 O

Ser Luell Tyr Luell Lell Wall Wall Trp Luell Trp Ser Ser His Ile Ala 115 12 O 125

Ser Gly Glin Pro Arg Lys Glu Asp Pro Luell Ala Lell Arg Luell 13 O 135 14 O

Wall Ile Ala His Asn Lell Phe Luell Luell Ser Lell Phe Met Cys 145 150 155 160

Wall Gly Luell Ile Ala Ala Ala Arg His Tyr Gly Ser Wall Trp Gly 1.65 17O 17s

Asn Arg Glu Arg Glu Pro Ala Met ASn Lell Lell Ile Wall 18O 185 19 O

Phe Met Ser Lys Lell Glu Phe Met Asp Thir Ala Ile Met Luell 195

Phe Arg Arg Asn Lell Arg Glin Wall Thir Luell His Wall His His 21 O 215 22O

Ala Ser Ile Ala Met Ile Trp Trp Ile Ile Cys Arg Phe Pro Gly 225 23 O 235 24 O

Ala Asp Ser Phe Ser Ala Ala Phe Asn Ser Ile His Wall Ala 245 250 255

Met Lieu Tyr Tyr Lieu 6. Ala Ala Thr Wall Ala Arg Asp Glu Lys 26 O 265 27 O

Arg Arg Arg Tyr Lell Phe Trp Gly Lell Thir Ile Ile Glin 285

Met Luell Glin Phe Lell Ser Phe Ile Gly Glin Ala Ile Ala Met Trp 29 O 295 3 OO

Lys Phe Glu Tyr Pro Gly Phe Gly Arg Met Lell Phe Phe Tyr 3. OS 310 315

Ser Wall Ser Luell Lell Ala Phe Phe Gly Asn Phe Phe Wall Lys Tyr 3.25 330 335

Ser Asn Ala Ser Glin Pro Thir Wall Wall Glu 34 O 345

<210s, SEQ ID NO 61 &211s LENGTH: 276 212. TYPE : PRT &213s ORGANISM: Thraulstochytrium sp. FJN-10

<4 OOs, SEQUENCE: 61 Met Asp Val Val Glu Glin Glin Trp Arg Arg Phe Wall Asp Ala Wall Asp 1. 5 1O 15

Asn Gly Ile Wall Glu Phe Met Glu His Glu Glu Pro Asn Lys Luell Asn 25 3O

Glu Gly Lys Luell Ser Thir Ser Thir Glu Glu Met Met Ala Luell Ile Wall 35 4 O 45

Gly Tyr Luell Ala Phe Wall Wall Luell Gly Ser Ala Phe Met Ala Phe SO 55 6 O

Wall Asp Pro Phe Glu Lell Phe Luell Lys Lell Wall His Asn Ile 65 70

Phe Luell Thir Gly Lell Ser Met Met Ala Thir Glu Ala Arg Glin 85 90 95 US 9,315,837 B2 113 114 - Continued

Ala Tyr Lieu. Gly Gly Luell Phe Gly ASn Pro Met Glu Lys Gly 1OO 105 11 O

Thir Glu Ser His Ala Pro Gly Met Ala Asn Ile Ile Tyr Ile Phe Tyr 115 12 O 125

Wall Ser Lys Phe Lieu. Glu Phe Luell Asp Thir Wall Phe Met Ile Luell Gly 13 O 135 14 O

Lys Lys Glin Lell Ser Phe Luell His Wall His His Ala Ser 145 150 155 160

Ile Ser Phe Ile Trp Gly Ile Ile Ala Arg Phe Ala Pro Gly Gly Asp 1.65 17s

Ala Tyr Phe Ser Thir Ile Lell Asn Ser Ser Wall His Wall Wall Luell Tyr 18O 185 19 O

Gly Ala Ser Thir Thir Luell Gly Thir Phe Met Arg Pro Luell 195

Arg Pro Tyr Ile Thr Thir Ile Glin Luell Thir Glin Phe Met Ala Met Wall 21 O 215 22O

Wall Glin Ser Val Tyr Asp Asn Pro Cys Asp Pro Glin Pro 225 23 O 235 24 O

Lell Val Lys Lieu. Luell Phe Trp Met Luell Thir Met Lell Gly Luell Phe 245 250 255

Gly Asn. Phe Phe Wall Glin Glin Luell Pro Ala Pro Lys 26 O 265 27 O

Glin Lys Thr Ile 27s

<210s, SEQ I D NO 62 &211s LENGT H: 12 212. TYPE : PRT ORGANISM: Articifical 22 Os. FEATU RE: &223s OTHER INFORMATION: Parietochloris incisa; Marchantia polymorpha; Ostreococcus tauri; Physcomitrella patens 22 Os. FEATU RE: <221 > NAMEA KEY: MISC FEATURE LOCATION: (3) . . (3) &223s OTHER INFORMATION: Xaa at position 3 may be any naturally occurring amino acid and up to one amino acid may be absent 22 Os. FEATU RE: <221 > NAMEA KEY: MISC FEATURE LOCATION: (6) . . (7) &223s OTHER INFORMATION: Xaa at positions 6-7 may be any naturally occurring amino acids and up to two of them may be absent 22 Os. FEATU RE: <221 > NAMEA KEY: MISC FEATURE LOCATION: (10) ... (10) <223> OTHER INFORMATION: Xaa at position 10 may be any naturally occurring amino acid and up to one amino acid may be absent

< 4 OOs SEQUENCE: 62 Phe Tyr Xaa Ser Lys Xaa Xaa Glu Phe Xaa Asp Thr 1. 5

SEQ ID NO 63 LENGTH: 13 212. TYPE : PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: Parietochloris incisa; Marchantia polymorpha; Ostreococcus tauri; Physcomitrella patens FEATURE: NAME/KEY: MISC FEATURE LOCATION: (2) .. (4) OTHER INFORMATION: Xaa at positions 2-4 may be any naturally occurring amino acids and up to three of them may be absent US 9,315,837 B2 115 116 - Continued

22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (11) . . (12) <223> OTHER INFORMATION: Xaa at positions 11-12 may be any naturally occurring amino acids and up to two of them may be absent <4 OOs, SEQUENCE: 63 Glin Xaa Xala Xala Lieu. His Val Tyr His His Xaa Xaa Ile 1. 5 1O

<210s, SEQ ID NO 64 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Parietochloris incisa; Marchantia polymorpha; Ostreococcus tauri; Physcomitrella patens 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (4) <223> OTHER INFORMATION: Xaa at positions 3-4 may be any naturally occurring amino acids and up to two of them may be absent 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Xaa at position 7 may be any naturally occurring amino acid and up to one amino acid may be absent 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Xaa at position 10 may be any naturally occurring amino acid and up to one amino acid may be absent

<4 OOs, SEQUENCE: 64 Asn Ser Xaa Xaa His Val Xaa Met Tyr Xaa Tyr Tyr 1. 5 1O

<210s, SEQ ID NO 65 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Parietochloris incisa; Marchantia polymorpha; Ostreococcus tauri; Physcomitrella patens 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (3) <223> OTHER INFORMATION: Xaa at positions 2-3 may be any naturally occurring amino acids and up to two of them may be absent 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: Xaa at positions 5-6 may be any naturally occurring amino acids and up to two of them may be absent <4 OOs, SEQUENCE: 65

Thr Xaa Xala Glin Xaa Xala Glin Phe 1. 5

The invention claimed is: claim 1, wherein said coding portion comprises a nucleic acid 1. A transgenic plant, a transgenic Seed, an alga trans sequence set forth in SEQID NO: 5. formed cell, a transfected or a transgenic alga, comprising a 4. The transgenic plant, the transgenic seed, the trans polynucleotide having a coding portion encoding a protein formed cell, the transfected alga or the transgenic alga of comprising the amino acid sequence set forth in any one of 60 claim 1, wherein said coding portion comprises a nucleic acid SEQID NOs: 1-3. sequence set forth in SEQID NO: 6. 2. The transgenic plant, the transgenic seed, the trans 5. A composition comprising the transgenic plant, the formed cell, the transfected alga or the transgenic alga of transgenic seed, the transformed cell, the transfected alga or claim 1, wherein said coding portion comprises a nucleic acid the transgenic alga of claim 1 and a carrier. sequence set forth in SEQID NO: 4. 6. The transgenic plant, the transgenic seed, the trans 3. The transgenic plant, the transgenic seed, the trans formed cell, the transfected alga or the transgenic alga of formed cell, the transfected alga or the transgenic alga of claim 1, wherein said polynucleotide having a coding portion US 9,315,837 B2 117 118 encoding a protein comprising the amino acid sequence set claim 1, grown under oleogenic conditions, under nitrogen forth in any one of SEQID NOs: 1-3 is an expression vector starvation conditions, or a combination thereof. comprising a coding portion encoding a protein comprising 10. The transgenic plant, the transgenic seed, the trans formed cell, the transfected alga or the transgenic alga of the amino acid sequence set forth in any one of SEQID NOs: claim 1, further comprising a polynucleotide having a coding 1-3. portion encoding a PUFA-specific elongase. 7. The transgenic plant, the transgenic seed, the trans 11. A method of producing very long-chain polyunsatu formed cell, the transfected alga or the transgenic alga of rated fatty acid (VLC-PUFA) comprising, making the trans claim 1, comprising linoleic acid (LA; 18:2006), C-linolenic genic plant, the transgenic seed, the transformed cell, the acid (ALA; 18:3(O3), oleic acid (18:1), dihomo-gamma-lino transfected alga or the transgenic alga of claim 1. lenic acid (20:3(O6), phosphatidylcholine (PC), diacylglyc 10 12. The method of claim 11, wherein the transgenic plant, eroltrimethylhomoserine (DGTS), phosphatidylethanola the transgenic seed, the transformed cell, the transfected alga mine (PE), or any combination thereof. or the transgenic alga is grown under oleogenic conditions, 8. The transgenic plant, the transgenic seed, the trans under nitrogen starvation conditions, or a combination formed cell, the transfected alga or the transgenic alga of thereof. claim 1, comprising eicosapentaenoic acid (EPA, 20:5c)3), 15 13. The method of claim 11, wherein said producing VLC docosahexaenoic acid (DHA, 22:603), dihomo-gamma-lino PUFA is enhancing oil storage, arachidonic acid accumula lenic acid (DGLA), arachidonic acid (ARA, 20:4()6), or any tion, eicosapentaenoic acid accumulation, docosahexaenoic combination thereof. acid accumulation, dihomo-gamma-linolenic acid accumula 9. The transgenic plant, the transgenic seed, the trans tion, or a combination thereof. formed cell, the transfected alga or the transgenic alga of k k k k k