US 2013 OO19341A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0019341 A1 Umidjon et al. (43) Pub. Date: Jan. 17, 2013

(54) DESATURASES OF A GREEN MICROALGA CI2N I/3 (2006.01) AND USES THEREOF CI2N 5/82 (2006.01) CI2P 7/64 (2006.01) (76) Inventors: Iskandarov Umidjon, Sde-Boker (IL); AOIH 5/00 (2006.01) Inna Khozin Goldberg, Sde-Boker (IL); AOIH 5/10 (2006.01) Zvi Hacohen, Omer (IL) CI2N 15/53 (2006.01) CI2N5/10 (2006.01) (21) Appl. No.: 13/520,607 (52) U.S. Cl. ... 800/281; 435/189: 536/23.2:435/320.1; (22) PCT Filed: Jan. 5, 2011 435/257.2:435/419:435/134; 800/298 e as (86). PCT No.: PCT/IL11AOOOO6 (57) ABSTRACT S371 (c)(1), Isolated proteins which are at least partially encoded by poly (2), (4) Date: Sep. 20, 2012 nucleotide sequences encoding novel desaturases are pro vided together with a composition which includes these iso Related U.S. Application Data lated proteins. A transgenic plant, a transgenic alga, or a (60) Provisional application No. 61/292,185, filed on Jan. transgenic seed transformed by the polynucleotides encoding 5, 2010. proteins which are at least partially encoded by novel desatu rases are also provided. The invention also includes a process Publication Classification for making a very long-chain polyunsaturated fatty acid in a transformed cell, a transgenic alga, or a transgenic plant (51) Int. Cl. expressing the isolated protein or proteins which are at least CI2N 9/02 (2006.01) partially encoded by the polynucleotide sequences encoding CI2N 15/63 (2006.01) novel A5, A6, or A12 desaturases.

Patent Application Publication Jan. 17, 2013 Sheet 6 of 16 US 2013/0019341 A1

Patent Application Publication Jan. 17, 2013 Sheet 8 of 16 US 2013/0019341 A1

Phaeodactylum tricornutum Thalassiosira pseudonana Pythium irregulare Mortierella alpina

C>Parietochloris incisa

90 Ceratodon purpureus a s gesatrasess

5. Marchantia polymorpha Mantoniella squamata Micromonas pusilla

Ostreococcus tauri

Ostreococcus lucimarinus

Phaeodactylum tricornutum O Pythium irregulare Phytophthora megasperma Dictyostelium discoideum Mortierella alpina S A desatara Ses Ostreococcus tauri

Mantoniella squamata

{d Parietochloris incisa Marchantia polymorpha

Physcomitrella patens S7 Chlorella vulgaris 88 K> Parietochloris incisa Chlamydomonas reinhardtii A desaturases 10 - Spinacia oleracea o Love hirsutum 38 Olea europaea Figure 2 Patent Application Publication Jan. 17, 2013 Sheet 9 of 16 US 2013/0019341 A1

exogenously supplied

(asa

2 4 2 Retention time (min)

FIGURE 3 Patent Application Publication Jan. 17, 2013 Sheet 10 of 16 US 2013/0019341 A1

100

-- PiDes12

80 -O-PiDess

- x - PiDeS5

- -A- - ARA 60 40

35 40 -

30

25

20 O 2 4. 6 8 10 12 14 Time (days)

FIGURE 4

Patent Application Publication Jan. 17, 2013 Sheet 13 of 16 US 2013/0019341 A1

o Strict Cutoff OOSe Cutoff ------

() r h O (9 0. o O op C an W. a on

O O 50 1OO 150 2OO 250 3OO Amino acid position

Figure 6 Patent Application Publication Jan. 17, 2013 Sheet 14 of 16 US 2013/0019341 A1

M. polymorpha ELO1 P. patens ELOI M. polymorpha ELO2 P. incisa ELOI O, tauri ELOl L. infantum ELO2 Thraustochytrium sp.FJN-10 ELOI M. alpina ELO1 P. salina ELOl O. tauri ELO2 T. pseudonana ELO2 C. intestinalis ELOI/2 X. tropicalis ELO2 O. mykiss R. norvegicus ELO2 H. sapiens ELO2 P. tricornutum ELOI T. pseudonana ELOI Thraustochytrium sp. FJN-10 ELO2 I. galbana ELO9

0.1

Figure 7 Patent Application Publication Jan. 17, 2013 Sheet 15 of 16 US 2013/0019341 A1

2

g

(d done g5 Sg so A. e f 3. re s pYES2+PiELO1

Figure 8A B. c.

pYES2+PiELO1 - pYES2 Retention tire Figure 8B Patent Application Publication Jan. 17, 2013 Sheet 16 of 16 US 2013/0019341 A1

2O 9 ; 16 O old 12

8

5 4 2 i o O 3 1 7 14 Time (days)

Figure 9 US 2013/0019341 A1 Jan. 17, 2013

DESATURASES OF A GREEN MICROALGA Isocrysis galbana and in the fresh water euglenophyte AND USES THEREOF Euglena gracilis, where LA and ALA are first elongated by C18 A9-specific fatty acid elongase followed by sequential FIELD OF INVENTION A8 and AS desaturations to ARA, DGLA or EPA. The extra plastidial A12 desaturase is an integral ER-bound protein 0001. This invention is directed to, interalia, proteins hav which is responsible for the desaturation of oleic acid and ing A2, A6, or A5 desaturase activity, isolated DNA molecules production of LA, mainly on phosphatidylcholine (PC). A5 encoding the same, and methods of making and utilizing the and A6 desaturases contain a fused cytochrome b5 domain in SaC. their N-terminus, serving as an electron donor, and introduce a double bond at a site closer to the carboxyl group than any BACKGROUND OF THE INVENTION of the pre-existing double bonds in the substrate fatty acid, 0002 Very long-chain polyunsaturated fatty acids (VLC thereby called front-end desaturases. Desaturases with A6 PUFA) of 20 or 22 carbon atoms are indispensable compo or A5 activity have been isolated from various organisms, nents of human nutrition. They are necessary for normal e.g., the nematode C. elegans, the fungus Mortierella alpina, life-long physiology and benefit the well-being of the human the moss Physcomitrella patens, the liverwort Marchantia body. Nutritionally important VLC-PUFAs include the polymorpha and the algae Phaeodactylum tricornutum, ()3-fatty acids, eicosapentaenoic acid (EPA, 20:5c)3) and Thalassiosira pseudonana and Ostreococcus tauri. Some of docosahexaenoic acid (DHA, 22:603) and the co6-fatty acid, these desaturases have been introduced together with PUFA arachidonic acid (ARA, 20:4()6) and dihomo-y-linolenic specific elongases into constructs for transformation of yeast acid (DGLA, 20:3(O6) which are the major components of and oil seed plants to reconstitute VLC-PUFA biosynthesis in membrane phospholipids of the retina, brain and testis. ARA the heterologous organisms. and DHA are the predominant fatty acids in the human brain and breast milk. ARA is necessary for normal fetal growth, SUMMARY OF THE INVENTION and cognitive development in infants. Many studies highly Suggested Supplementation of infant formula with DHA and 0006. In one embodiment, the present invention provides ARA. Besides the structural function in membranes, ARA is an isolated protein comprising, an amino acid sequence set the primary Substrate in eicosanoids biosynthesis which regu forth in SEQID NO: 1. lates many physiological processes Such as homeostasis, 0007. In another embodiment, the present invention fur reproduction, immune and inflammatory responses. ther provides an isolated protein comprising, an amino acid 0003 Microalgae are the most efficient producers and one sequence set forth in SEQID NO: 2. of the richest sources of VLC-PUFAs. Furthermore, algae can 0008. In another embodiment, the present invention fur be used as sources of genes for the implementation of VLC ther provides an isolated protein comprising, an amino acid PUFA biosynthesis in genetically engineered oil crops. The sequence set forth in SEQID NO:3. genetic information on enzymes involved in the biosynthesis of VLC-PUFA in some algae led to in vivo applications of 0009. In another embodiment, the present invention fur VLC-PUFA production in seed oil. The gene pool of the green ther provides a composition comprising a protein comprising, freshwater microalga Parietochloris incisa (Trebouxio an amino acid sequence set forth in SEQ ID NO: 1, a com phyceae) is of special interest since it is the only known position comprising a protein comprising, an amino acid microalga able to accumulate extraordinary high amounts of sequence set forth in SEQID NO: 2, a composition compris ARA-rich triacylglycerols (TAG). When P incisa is culti ing a protein comprising, an amino acid sequence set forth in vated under nitrogen starvation, the condition triggering Stor SEQID NO:3, or a composition comprising any combination age oil accumulation, ARA constitutes about 60 percent of thereof. total fatty acids (TFA) and over 95 percent of cellular ARA is 0010. In another embodiment, the present invention fur deposited in TAG in cytoplasmic lipid bodies. ther provides a transgenic plant, a transgenic seed, a trans 0004. The biosynthesis of VLC-PUFA in microalgae fol formed cell, or a transgenic alga transformed by a polynucle lows two major pathways, designated as (D6 and (03. In these otide encoding: (1) a protein comprising, an amino acid pathways, linoleic acid (LA; 18:2()6) and O-linolenic acid sequence set forth in SEQID NO: 1, (2) a protein comprising, (ALA; 18:303) go through sequential. A6 desaturation, A6 an amino acid sequence set forth in SEQ ID NO: 2, (3) a elongation and A5 desaturation, yielding ARA and EPA, protein comprising, an amino acid sequence set forth in SEQ respectively. E.g., in the red microalga Porphyridium cruen ID NO:3, or a transgenic plant, a transgenic seed, or a trans tum and the green microalga P incisa, oleic acid (18:1) is first genic alga transformed by any combination of the polynucle desaturated to LA and Y-linolenic acid (GLA, 18:3(O6) otides (1), (2), and (3). through A12 and A6 desaturations, followed by elongation to 0011. In another embodiment, the present invention fur 20:3 (D6 and A5 desaturation to yield ARA via the ()6 pathway. ther provides a method of producing very long-chain polyun In P incisa, the extraplastidial lipids, phosphatidylcholine saturated fatty acid (VLC-PUFA) in a plant, a plant cell, or an (PC) and the betaine lipid, diacylglyceroltrimethylho alga comprising the step of transforming a plant, an alga, or a moserine (DGTS), are involved in the A12 and, subsequently, plant cell with a polynucleotide encoding: (1) a protein com the A6 desaturations, whereas phosphatidylethanolamine prising, an amino acid sequence set forth in SEQID NO: 1. (PE) along with PC are the suggested major substrates for the (2) a protein comprising, an amino acid sequence set forth in A5 desaturation of 20:3()6 to 20:4()6. The same enzymes are SEQ ID NO: 2, (3) a protein comprising, an amino acid involved in the biosynthesis of VLC-PUFA through the co3 sequence set forth in SEQID NO:3, or transforming a plant, pathway in the green microalga Ostreococcus tauri. a plant cell, oran alga with any combination of the polynucle 0005 VLC-PUFAs may also be generated by an alterna otides (1), (2), and (3), thereby producing a VLC-PUFA in a tive A8 desaturation pathway. E.g., in the marine haptophyte plant, a plant cell, or an alga. US 2013/0019341 A1 Jan. 17, 2013

BRIEF DESCRIPTION OF THE DRAWINGS (A6, Pincisa), AAV67797 (A6, O. tauri), AAV67798 (A5, O. 0012 FIG. 1. Depicts the deduced amino acid sequences tauri), AAT85662 (A6, M. polymorpha), BAE71129 (A5, M. of P incisa PiDes12 (A), PiDesG (B), and PiDes5 (C) are polymorpha), AAL84174 (A6, P. patens), CAJ 30819 (A6, aligned with their closest homologs using CLUSTAL W Thraustochytrium sp.), CAM55873 (A5, Thraustochytrium (1.83) multiple sequence alignment program (default). Con sp.), AAF70417 (A6, M. alpina), XP 001467802 (L. infan served motifs characteristic of each desaturase sequence are tum), AAV67803 (A6/A5, O. mykiss), NP 001029014 (A6/ highlighted. GeneBank accession numbers for the sequences A5, C. intestinalis), NP 068586 (A6/A5, H. sapiens), are: A) C. reinhardtii (XP 001691669); C. vulgaris AAY15135 (A5, P. salina), CAM55851 (A6P tricornutum), (BAB78716), G. hirsutum (AAL37484), S. oleracea AAL37626 (A9, I. galbana), AAV67799 (A6, T. pseud (BAC22091), O. europaea (AAW63040). B) M. polymorpha 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 (CAQ30479). C) O. tauri (CAL57370), M. squamata (0019 FIG.8. Isa GC plot of FAMES of recombinant yeast (CAQ30478), M. polymorpha (AAT85663), D. discoideum harboring pYES2 and PiELO1 fed with 18:306 (A) and (BAA37090), M. alpina (AAC72755), P tricornutum 18:403 (B) CONTROL. (AYO82392). 0020 FIG. 9. Is a bar graph summarizing the results of 0013 FIG. 2. Is an unrooted phylogram of PiDes 12, quantitative Real-time RT-PCR analysis of PiELO1 gene PiDesG. PiDes5 and some functionally characterized A12, A6 expression in log phase (Time 0) and N-starved (3, 7 & 14 d) and A5 desaturases (vertebrate and invertebrate desaturases are not included). The alignment was generated by the cells of P incisa. The transcript abundance of the gene was CLUSTALW program and the unrooted phylogram was con normalized to 18S SSU rRNA gene. structed in the neighbor-joining method using the MEGA4 software 47. GeneBank sources of the sequences are: DETAILED DESCRIPTION OF THE INVENTION BAB78716 (A12, Chlorella vulgaris), XP 001691669 (A12, C. reinhardtii), BAC22091 (A12, Spinacia oleracea), 0021. In one embodiment, the present invention provides AAL37484 (A12, Gossypium hirsutum), AAW63040 (A12, an isolated protein. In another embodiment, the present Olea europaea), CAB94993 (A6, Ceratodon purpureus), invention provides that the isolated protein is a polypeptide. AAT85661 (A6, M. polymorpha), BAA85588 (A6, M. In another embodiment, the present invention provides that alpina), AAL92563 (A6, P. tricornutum), AAX14505 (A6, T. the isolated protein is an enzyme. In another embodiment, the pseudonana), (A6, Pythium irregulare), CAL57370 (A5, O. present invention provides that the isolated protein is a tauri), AAT85663 (A5, M. polymorpha), AAL13311 (A5, P desaturase. In another embodiment, the present invention irregulare), CAD53323 (A5, Phytophthora megasperma), provides that the isolated protein is an algal desaturase. In BAA37090 (A5, Dictyostelium discoideum), AAC72755 (A5, another embodiment, the present invention provides that the M. alpina), CAQ30478 (A5, M. squamata), CAQ30479 (A6, isolated protein is a microalgae desaturase. In another M. squamata), AAW70 159 (A6, O. tauri), CS020055 (A5, P embodiment, the present invention provides that the isolated patens). protein is a 0014 FIG.3. Provides graphs representing GCFAMEs of recombinant yeast harboring pYES2 (control), pY PiDesG 0022 A12 desaturase. In another embodiment, the present (A) fed with 18:2 or 18:303, and pYPiDes5 (B) fed with invention provides that the isolated protein is a A6 desaturase. 20:306, 20:4c)3 or 18:1. In another embodiment, the present invention provides that 0015 FIG.4. Is a graph showing the changes in expression the isolated protein is a A5 desaturase. In another embodi of the PiDes 12, PiDes6, and PiDes5 genes under N-starvation ment, the present invention provides that the isolated protein and ARA percent share in total fatty acids. The transcript is a microalgae desaturase produced in a plant cell. In another abundance of the genes was normalized to that of the actin embodiment, the present invention provides that the isolated gene. protein is a microalgae desaturase produced in an algal cell. 0016 FIG. 5. Depicts the amino acid sequence of P incisa PiELO1 aligned with its closest homologs using CLUSTAL 0023. In another embodiment, the present invention pro W (1.83) multiple sequence alignment program (default). vides a A12 desaturase comprising the amino acid sequence: Conserved motifs characteristic of PUFA elongase sequences are highlighted. GeneBank accession numbers for the sequences are OtBLO1 (O. tauri, AAV67797), MpeLO1 (M. (SEQ ID NO: 1) polymorpha, AAT85662), PpELO1 (P. patens, AAL84174), MGKGGCYOAGPPSAKKWESRVPTAKPEFTIGTLRKAIPVHCFERSIPRSF MpELO2 (M. polymorpha, BAE71129), and ThrELO1 AYLAADLAAIAWMYYLSTFIDHPAVPRVLAWGLLWPAYWYFOGAVATGVW Thraustochytrium sp. FIN-10, ABC18313). 0017 FIG. 6. Is a hydropathy plot of the amino acid VIAHECGHQAFSPYOWLNDAVGLVLHSCLLVPYYSWKHSHRRHHSNTGST sequence of PiELO1. The lower dashed line and the upper TKDEVFWPREAAMVESDFSLMOTAPARFLVIFWSLTAGWPAYLFANASGR line represent the loose transmembrane region cutoff and the strict transmembrane region cutoff, respectively. KYGKWANHFDPYSPIFTKRERSEIWWSDWALTWWIAGLYSLGKAFGWAWL 0018 FIG. 7. Is an unrooted phylogram of PiELO1 and some other functionally characterized PUFA elongases. The WKEYVIPYLIVNMWLVMITLLOHTHPELPHYADKEWDWLRGALATCDRSY alignment was generated by the CLUSTALW program and GGMPDHLHHHIADTHVAHHLFSTMPHYHAOEATEAIKPILGKYYKODKRN the unrooted phylogram was constructed by the neighbor joining method using the MEGA4 software. GeneBank WWAALWEDFSLCRYWAPDTAGSGILWFRA accession numbers for the PUFA elongases are: ACK99719 US 2013/0019341 A1 Jan. 17, 2013

0024. In another embodiment, the present invention pro ment, the desaturase comprises an amino acid sequence that is vides a A6 desaturase comprising the amino acid sequence: at least 98% homologous to the amino acid sequence of SEQ ID NO: 1, SEQID NO: 2, or SEQ ID NO:3. (SEQ ID NO: 2) 0028. In another embodiment, the desaturase of the MCOGOAWOGLRRRSSFLKLTGDAIKGAVAAISDFNKLPAATPWFARRSLS present invention comprises an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID DSALOORDGPRSKOOVTLEELAOHNTPEDCWLVIKNKVYDVSGWGPOHPG NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi GHVIYTYAGKDATDVFACFHAOTTWSOLRPFCIGDIVEEEPMPALLKDFR ment, the desaturase comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQID ELRTRLOOOGLFRSNKLYYLYKVASTLSLLAAALAVLITORDSWLGLVGG NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi AFLLGLFWOOSGWLAHDFLHHOVFTDROWNNWMGYFLGNVCOGFSTDWWK ment, the desaturase comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQID SKHNWHHAWPNELDSDSKAARDPDIDTLPLLAWSSEMLDSMSNSGARLFW NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi RMOHYFFFPILLFARMSWCOOSVAHASDLSRTSKAGVYELAYLALHYAWF ment, the desaturase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQID LGAAFSVLPPLKAVWFALLSOMFSGFLLSIVFVOSHNGMEWYSDTKDFWT NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi AOIVSTRDILSNVWNDWFTGGLNYOIEHHLFPTLPRHNLGKVOKSIMELC ment, the desaturase comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQID HKHGLVYENCGMATGTYRVLORLANVAAEA. NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi ment, the desaturase comprises an amino acid sequence that is 0025. In another embodiment, the present invention pro at least 90% identical to the amino acid sequence of SEQID vides a AS desaturase comprising the amino acid sequence: NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi ment, the desaturase comprises an amino acid sequence that is (SEQ ID NO : 3) at least 95% identical to the amino acid sequence of SEQID MMAVTEGAGGVTAEVGLHKRSSOPRPAAPRSKLFTLDEVAKHDSPTDCWV NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi ment, the desaturase comprises an amino acid sequence that is VIRRRVYDWTAWVPOHPGGNLIFVKAGRDCTOLFDSYHPLSARAVLDKFY at least 98% identical to the amino acid sequence of SEQID IGEVDVRPGDEOFLVAFEEDTEEGOFYTVLKKRVEKYFRENKLNPRATGA NO: 1, SEQID NO: 2, or SEQID NO:3. 0029. In another embodiment, the desaturase as described MYAKSLTILAGLALSFYGTFFAFSSAPASLLSAWLIGICMAEWGWSIMHD herein comprises at least a portion of the amino acid shown in ANHGAFARNTWASHALGATLDIVGASSFMWRQOHV VGHHAYTNVDGODPD SEQID. NO: 1, SEQID NO: 2, or SEQID NO:3. In another embodiment, the desaturase as described herein is a variant of LRVKDPDWRRVTKFQPQOSYQAYOHIYLAFLYGLLAIKSWLLDDFMALSS SEQID. NO: 1, SEQID NO: 2, or SEQID NO:3. In another embodiment, the term “variant' in relation to a certain GAIGSWKWAKLTPGEKLWFWGGKALWLGYFWLLPWWKSRHSWPLLAACWL sequence means a protein or a polypeptide which is derived LSEFWTGWMLAFMFOVAHVTSDVSYLEADKTGKVPRGWAAACAATTADFA from the sequence through the insertion or deletion of one or more amino acid residues or the Substitution of one or more HGSWFWTOISGGLNYOVWHHLFPGICHLHYPAIAPIVLDTCKEFNVPYHV amino acid residues with amino acid residues having similar YPTFWRALAAHFKHLKDMGAPTAIPSLATWG: properties, e.g., the replacement of a polar amino acid residue with another polar amino acid residue, or the replacement of 0026. In another embodiment, the desaturase of the a non-polar amino acid residue with another non-polar amino present invention comprises an amino acid sequence that is at acid residue. In all cases, variants must have a desaturase least 60% homologous to the amino acid sequence of SEQID function as defined herein. NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi ment, the desaturase comprises an amino acid sequence that is 0030. In another embodiment, the desaturase as described at least 70% homologous to the amino acid sequence of SEQ hereinfurther comprises a leader peptide. In another embodi ID NO: 1, SEQ NO:2, or SEQID NO:3. In another embodi ment, the leader peptide allows the polypeptide to be specifi ment, the desaturase comprises an amino acid sequence that is cally located or targeted to a target organelle within the cell. at least 75% homologous to the amino acid sequence of SEQ In another embodiment, the desaturase as described herein ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In another further comprises a sequence motif responsible for microso embodiment, the desaturase comprises an amino acid mal localization. In another embodiment, a desaturase as sequence that is at least 80% homologous to the amino acid described herein further comprises chemical modification sequence of SEQID NO: 1, SEQID NO: 2, or SEQID NO: Such as glycosylation that increases its stability. In another 3. In another embodiment, the desaturase comprises an amino embodiment, a desaturase as described herein further com acid sequence that is at least 85% homologous to the amino prises a peptide unrelated to desaturase which increases its acid sequence of SEQID NO: 1, SEQID NO: 2, or SEQID stability. NO:3. In another embodiment, the desaturase comprises an 0031. In another embodiment, the present invention pro amino acid sequence that is at least 90% homologous to the vides an isolated PUFA desaturase. In another embodiment, amino acid sequence of SEQID NO: 1, SEQID the present invention provides an isolated polypeptide com 0027 NO: 2, or SEQ ID NO:3. In another embodiment, prising a functional long chain polyunsaturated fatty acid the desaturase comprises an amino acid sequence that is at (PUFA) desaturase. In another embodiment, the present least 95% homologous to the amino acid sequence of SEQID invention provides that the polypeptide has the function of NO: 1, SEQID NO:2, or SEQID NO:3. In another embodi desaturating a chain longer than 18 carbons fatty acid. In US 2013/0019341 A1 Jan. 17, 2013

another embodiment, the present invention provides that the relates with the production of DGLA precursors, EPA pre polypeptide has the function of desaturating a chain longer cursors, DHA precursors, ARA precursors, or any combina than 20 carbons fatty acid. tion thereof. 0032. In another embodiment, the present invention pro 0036. In another embodiment, the present invention pro vides an isolated PUFA desaturase comprising a fused N-ter vides an isolated polynucleotide encoding the protein as minal cytochrome b5 domain. In another embodiment, the described herein. In another embodiment, an isolated poly present invention provides an isolated PUFA desaturase nucleotide is an isolated DNA molecule. In another embodi which desaturates (06 substrates. In another embodiment, the ment, an isolated polynucleotide is an isolated cDNA mol present invention provides an isolated PUFA desaturase ecule. In another embodiment, the isolated polynucleotide which desaturates both (O3 substrates. In another embodi comprises a sequence encoding the protein as described ment, the present invention provides an isolated PUFA herein. In another embodiment, the isolated polynucleotide desaturase which desaturates both (03 and (06 substrates. In comprises a DNA sequence encoding a desaturase as another embodiment, the present invention provides an iso described herein. In another embodiment, the isolated poly lated PUFA desaturase encoded by SEQID NO: 1 (PiDes 12 nucleotide comprises a DNA sequence encoding a polypep or A12). In another embodiment, the present invention pro tide comprising a desaturase activity. In another embodiment, vides an isolated PUFA desaturase encoded by SEQID NO: the isolated polynucleotide comprises a DNA sequence 2 (PiDesG or A6). In another embodiment, the present inven encoding a polypeptide consisting a desaturase activity. tion provides an isolated PUFA desaturase encoded by SEQ ID NO: 3 (PiDes5 or A5). In another embodiment, the sub 0037. In another embodiment, the isolated polynucleotide strate for the present invention isolated PUFA desaturase is comprises a DNA sequence comprising the sequence: 18:2006, 20:306, 20:4c03, and 20:303 0033. In another embodiment, the present invention pro (SEQ ID NO: 4, PiDes12) vides an isolated PUFA desaturase which desaturates 20:303 atggggaaaggaggctgttaccaggc.cgggcctic ctago.gcaaagaaatg to a non-methylene-interrupted 20:4. In another embodi ment, the present invention provides that PiDes5 desaturates ggagagtagggtgcc cactgccaaac cc.gagttcacgat.cggalaccCtcC 20:3c)3 to a non-methylene-interrupted 20:4. In another gcaaagctat accogg to cactgctt.cgaacggtc. catcc ct cqgt cattg embodiment, the present invention provides that PiDes5 con verts 20:4c03 into the respective A5 product, 20:5c)3 (EPA) as CctaccttgcggCagacCtggcggct attgcggit Catgtactacctgagc well as the added 18:1 into the non-methylene-interrupted actitt catcgatcatc.ccgc.cgtgcc.gcgggtc.ctggcctggggtttgct 18:2A59. 0034. In another embodiment, the present invention pro gtggcCtgcc tactggtact tccalaggtgctgtggcgacaggcgtctggg vides a protein comprising a desaturase activity. In another tgattgct cacgagtgcggccaccaggcgttcticgc.cctaccagtggctic embodiment, the present invention provides a protein consist ing a desaturase activity. In another embodiment, the present aacgacgctgtggggcttgttgctgcact Cotgcttgctggtgc cct atta invention provides that the protein of the invention is a recom ct cotggaag cact cacacagacggcaccact coaacaccggaagicacca binant desaturase. In another embodiment, the present inven tion provides that the desaturase is a polyunsaturated fatty ccaaggatgaggtgtttgtc.ccc.cgggalagcagc catggtggagt cqgac acid (PUFA)-specific desaturase. In another embodiment, the present invention provides that the desaturase desaturates ttct cottgatgcagacagct cocqcgcggttcc tiggtcatctitcgt.ctic precursors of arachidonic acid. In another embodiment, the gctgaccgctggctggcctgcctacctgtttgccaatgcatctggcc.gca present invention provides that the desaturase desaturates precursors of EPA. In another embodiment, the present inven agtatggcaa.gtgggccaaccactittgacc cc tact cacccatctt cacc tion provides that the desaturase desaturates immediate pre aa.gc.gc.gagcgcago.gagat.cgttgtcagcgatgtc.gc.gctgacggtggit cursors of arachidonic acid (ARA). In another embodiment, the present invention provides that the protein as described catcgcggggctic tact cqctgggcaaggcgtttggctgggcctggctgg herein is used to elevate PUFA levels in animals, thereby providing a ready source of PUFAs. tdaaggagtatgtgatc.ccctacct catcgtcaa.catgtggctggtcatg 0035. In another embodiment, the expression and/or tran at Cacgctgctgcagcacacgcaccc.cgagctg.ccgcactacgc.cgacaa Scription of the desaturase as described herein is up-regulated ggagtgggactggctg.cgcggcgc.gctggccacctg.cgatcgcagctacg during nitrogen starvation. In another embodiment, the expression and/or transcription of the desaturase as described gcgg catgc.cggaccacctgcaccacca catcgc.cgacacgcacgtc.gct herein is up-regulated under oleogenic conditions. In another Caccacctgttct coac catgcc.gcactaccatgcgcaggaggcgactga embodiment, oleogenic conditions comprise the presence of a A6 substrate for A6 or A5 fatty acid desaturase. In another ggcgat Caagcc catcctgggcaagtact acaag Cagga caag.cgcaacg embodiment, oleogenic conditions comprise 18:2006 and 20:3(O6. In another embodiment, oleogenic conditions com totgggcagcgct Ctgggaggattt Cagcctgtgcc.gctatgtggcgc.ct prise nitrogen starvation. In another embodiment, the expres gacacagcaggct cqggcatcCtgtggttcc.gc.gcttga. In sion and/or transcription level of the desaturases as described herein correlates with the production of ARA precursors. In another embodiment, SEQ ID NO: 4 encodes the amino another embodiment, oleogenic conditions comprise nitro gen starvation. In another embodiment, the expression and/or acid sequence of SEQ ID NO: 1. transcription level of the desaturases as described herein cor

US 2013/0019341 A1 Jan. 17, 2013

isolated polynucleotide comprises a DNA sequence that is at or substrates that are involved in the biosynthesis of VLC least 90% homologous to the nucleic acid sequence of SEQ PUFA. In another embodiment, the present invention com ID NO:4, SEQID NO:5, SEQID NO: 6, or SEQID NO:8. prises an algal desaturase or a nucleic acid molecule encoding In another embodiment, the isolated polynucleotide com the same combined with additional algal proteins and/or prises a DNA sequence that is at least 95% homologous to the enzymes and/or substrates that are involved in the biosynthe nucleic acid sequence of SEQID NO: 4, SEQID NO. 5, SEQ sis of VLC-PUFA. In another embodiment, the present inven ID NO: 6, or SEQ ID NO: 8. In another embodiment, the tion provides that the alga is a microalga. In another embodi isolated polynucleotide comprises a DNA sequence that is at ment, the present invention comprises a microalgae least 98% homologous to the nucleic acid sequence of SEQ desaturase or a nucleic acid molecule encoding the same ID NO:4, SEQID NO:5, SEQID NO: 6, or SEQID NO:8. combined with additional microalgae proteins and/or 0041. In another embodiment, the isolated polynucleotide enzymes and/or substrates that are involved in the biosynthe comprises a DNA sequence that is at least 60% identical to the SiS of VLC-PUFA. nucleic acid sequence of SEQID NO:4, SEQID NO: 5, SEQ 0044. In another embodiment, the present invention pro ID NO: 6, or SEQ ID NO: 8. In another embodiment, the vides that algae proteins comprise an elongase. In another isolated polynucleotide comprises a DNA sequence that is at embodiment an elongase is described in PCT/IL2009/001117 least 70% identical to the nucleic acid sequence of SEQ ID which is hereby incorporated in its entirety by reference. In NO: 4, SEQID NO: 5, SEQID NO: 6, or SEQID NO:8. In another embodiment, the present invention provides that another embodiment, the isolated polynucleotide comprises a microalgae proteins comprise the P incisa PiELO1 gene DNA sequence that is at least 75% identical to the nucleic acid product. In another embodiment, the present invention pro sequence of SEQID NO: 4, SEQID NO:5, SEQID NO: 6, or vides that elongase as described herein comprises the amino SEQ ID NO: 8. In another embodiment, the isolated poly acid sequence: nucleotide comprises a DNA sequence that is at least 80% identical to the nucleic acid sequence of SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In another (SEO ID NO: 7) embodiment, the isolated polynucleotide comprises a DNA MALTAAWHKYDAIVSRFWFDGLRRVGLOEIOGHPSVITAHLPFIASPTPO sequence that is at least 85% identical to the nucleic acid WTFWLAYLLIVWCGVAALRTRKSSAPREDPAWLRLLVOAHNLVLISLSAY sequence of SEQID NO: 4, SEQID NO:5, SEQID NO: 6, or SEQ ID NO: 8. In another embodiment, the isolated poly MSSAACYYAWKYGYRFWGTNYSPKERDMGGLIYTFYWSKLYEFWD TLIML nucleotide comprises a DNA sequence that is at least 90% identical to the nucleic acid sequence of SEQID NO: 4, SEQ LKGKVEQVSFLHWYHHASISTIWWAIAYWAPGGDAWYCCFLNSLVHVLMY ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In another TYYLLATLLGKDAKARRKYLWWGRYLTOFOMFOFWTMMLEAAYTWAYSPY embodiment, the isolated polynucleotide comprises a DNA sequence that is at least 95% identical to the nucleic acid PKFLSKLLFFYMITLLALFANFYAOKHGSSRAAKOKLQ. sequence of SEQID NO: 4, SEQID NO:5, SEQID NO: 6, or SEQ ID NO: 8. In another embodiment, the isolated poly 0045. In another embodiment, the elongase as described nucleotide comprises a DNA sequence that is at least 98% herein id encoded by a polynucleotide comprising a DNA identical to the nucleic acid sequence of SEQID NO: 4, SEQ sequence comprising the sequence: ID NO. 5, SEQ NO: 6, or SEQID NO: 8. 0042. In another embodiment, the present invention com (SEQ ID NO: 8) prises a desaturase or a nucleic acid molecule encoding the atggcattgacggcggcctgg cacaagtacgacgct atcgittagt cqctt same combined with additional proteins and/or enzymes and/ or substrates that are involved in the biosynthesis of VLC tgttitt catggcttgcgcagggttggcctgcaa.gagattcaaggccacc PUFA. In another embodiment, the present invention com cct cqgtgat caccgc.ccacct tcc.ctt catago ct coccaacgc.cacaa prises a composition comprising a desaturase or a nucleic acid molecule encoding the same combined with additional gtgacgttcgtgctggcct atctgctgattgttgtctg.cggggttgcc.gc proteins and/or enzymes and/or substrates that are involved in the biosynthesis of VLC-PUFA. In another embodiment, the totgcgtacgagaaagttctgtc.cgcacct Cogaggat.ccggcgtggctgc present invention comprises a transgenic plant comprising a gactgcttgttgcaa.gc.gcaca acttggtgctaat cagccttagcgc.ctac desaturase or a nucleic acid molecule encoding the same combined with additional proteins and/or enzymes and/or atgtcc totgcc.gc.ctgctactatoctitggaaatacggctataggittittg substrates that are involved in the biosynthesis of VLC gggcacaaactatagc.cccaaggagcggga catgggagggct Catctata PUFA. In another embodiment, the present invention com prises a transgenic alga comprising a desaturase or a nucleic CcttttacgtgtcCaagctgtacgagtttgttggatacgctgat catgctg acid molecule encoding the same combined with additional proteins and/or enzymes and/or substrates that are involved in ct Caagggcaaggtggagcaggttt Ctttitttgcacgt.ctaccaccacgc the biosynthesis of VLC-PUFA. In another embodiment, the tt CC at at CC acgatctggtgggcaatcgcatacgt.cgcacctggtggtg present invention comprises a transfected or a transformed cell comprising a desaturase or a nucleic acid molecule acgc.ctgg tactgctgctt cotgaacticgctggit coacgtact catgitac encoding the same combined with additional proteins and/or acatactacctgcttgcgacgctgctgggaaaggacgc.caaggcgcggcg enzymes and/or substrates that are involved in the biosynthe SiS of VLC-PUFA. Caagtatttgttggtggggacgctacct cacticagttccagatgttcCagt 0043. In another embodiment, the present invention com ttgttgacgatgatgctic gaggcagcgtacacttgggcct actict c cctac prises a desaturase or a nucleic acid molecule encoding the same combined with additional proteins and/or enzymes and/ US 2013/0019341 A1 Jan. 17, 2013

- Continued the combination of A5, A6, and/or A12 desaturases and either cc caagtttittatcaaagctgctgttatttaCatgat cactctgttggcc c)3 or ()6 C18 substrates yields DGLA, ARA, DHA and/or EPA Ctgtttgcaaacttictatgcacagaag catggcagcagc.cgggcagc.cala 0049. In another embodiment, the invention provides con gcaaaagctgcagtaa. junction of P incisa A12, A6, and A5 desaturases, which are set of P incisa genes involved in the biosynthesis of ARA. In 0046. In another embodiment, the present invention pro another embodiment, the invention provides conjunction of P vides a composition comprising a desaturase as described incisa A12, A6, and A5 desaturase and A6 specific PUFA herein. In another embodiment, the present invention pro elongase (as described herein), which are set of P incisa vides a composition comprising the desaturase as described genes involved in the biosynthesis of DGLA, ARA, DHA. herein and a VLC-PUFA elongase. In another embodiment, EPA, or any combination thereof. the present invention provides a composition comprising a 0050. In another embodiment, a desaturase as described protein as described herein. In another embodiment, the herein comprises three histidine rich motifs (his-boxes). In present invention provides a composition comprising the another embodiment, A6 (PiDesG) and A5 (PiDes5) desatu polynucleotide as described herein. In another embodiment, rases comprise fused cytochrome b5 at their N-terminus, the present invention provides a composition comprising a Supporting their microsomal localization. In another embodi polynucleotide encoding an elongase and the polynucleotide ment, A6 (PiDesG) and A5 (PiDes5) desaturases comprise a as described herein. In another embodiment, the present HPGG quartet along with four amino acids conserved in all invention provides a composition comprising a vector com cytochrome b5 fusion desaturases (FIG. 1). prising the polynucleotide as described herein. In another 0051. In another embodiment, transforming a plant with embodiment, the present invention provides a composition an algal-derived gene Such as described herein produces bet comprising a vector comprising a polynucleotide encoding an terresults in comparison to fungal genes. In another embodi elongase and a polynucleotide as described herein. In another ment, transforming a plant with an algal-derived gene Such as embodiment, the present invention provides a composition described herein in combination with additional genes that comprising a combination of vectors which comprise poly encode proteins that are involved in the biosynthesis of VLC nucleotides encoding an elongase and polynucleotides PUFA produces better results in comparison to fungal or encoding desaturases. In another embodiment, a composition wild-type genes. In another embodiment, transforming a Such as described herein comprises a carrier. In another plant with an algal-derived gene Such as described herein embodiment, a carrier stabilizes a protein or a nucleic acid (desaturase) in combination with an elongase as described molecule of the invention. In another embodiment, one of herein produces better results in comparison to fungal or skill in the art will readily identify a known suitable carrier to wild-type genes. In another embodiment, transforming a be used with the composition as described herein. In another plant with a combination of algal-derived genes such as embodiment, a carrier is a buffer such as but not limited to a described herein produces better results (such as ARA pro phosphate buffer. duction) in comparison to fungal or wild-type genes. In another embodiment, transforming a plant with a combina 0047. In another embodiment, one of skill in the art is able tion of algal-derived desaturase genes such as described to prepare a composition comprising a desaturase as herein produces better results (such as ARA production) in described herein. In another embodiment, one of skill in the comparison to fungal or wild-type genes. In another embodi artisable to prepare a composition comprising a combination ment, P incisa is the richest plant source of ARA. In another of elongases and desaturases as described herein. In another embodiment, P incisa is the richest algal source of ARA. In embodiment, one of skill in the art is able to prepare a com another embodiment, algal-derived genes such as described position comprising a polynucleotide as described herein. In herein are more effective alone or in combination than those another embodiment, one of skill in the art is able to prepare of other sources. a composition comprising a combination of polynucleotides, 0052. In another embodiment, algae as described herein plasmids, vectors etc. as described herein. In another embodi are eukaryotic organisms. In another embodiment, algae as ment, the present invention provides a composition compris described herein are photoautotrophic. In another embodi ing the protein as described herein to be used in foodstuffs, ment, algae as described herein are mixotrophic. In another dietary Supplements or pharmaceutical compositions. In embodiment, algae as described herein are unicellular. In another embodiment, the present invention provides a com another embodiment, algae as described herein are multicel position comprising the proteinas described herein to be used lular. In another embodiment, algae as described herein are in industrial applications for the manufacturing of VLC-PU Excavata algae. In another embodiment, algae as described FAS. In another embodiment, the present invention provides a herein are Rhizaria algae. In another embodiment, algae as composition comprising the VLC-PUFAs, the products of the described herein are Chromista algae. In another embodi enzymes of the present invention. In another embodiment, a ment, algae as described herein are Alveolata algae. composition comprising VLC-PUFAs is used in foodstuffs, 0053. In another embodiment, an algal gene and protein of dietary Supplements or pharmaceutical compositions. the present invention is Superior when compared to its homo 0.048. In another embodiment, the invention includes a logues with respect to efficient production of PUFAs. In combination of A5, A6, and/or A12 desaturases. In another another embodiment, transforming a first alga with an algal embodiment, the invention includes a composition compris gene derived from a second alga Such as described herein ing the combination of A5, A6, and/or A12 desaturases. In produces better results in comparison to fungal genes. In another embodiment, the invention includes a composition another embodiment, a second algal gene is a gene as comprising the combination of A5, A6, and/or A12 desatu described herein. In another embodiment, a first and a second rases and either ()3 or ()6 C18 substrates. In another embodi algal are of different species. In another embodiment, trans ment, the invention provides that a composition comprising forming a first alga with an algal gene derived from a second US 2013/0019341 A1 Jan. 17, 2013

alga Such as described herein in combination with additional embodiment, a plant is an oil crop. In another embodiment, genes that encode proteins that are involved in the biosynthe the transformed plant is an oil crop. sis of VLC-PUFA produces better results in comparison to 0057. In another embodiment, the present invention pro fungal or wild-type genes. In another embodiment, trans vides a transgenic plant, a transgenic seed, or a transgenic forming an alga with an algal gene (Such as desaturase) alga transformed by a polynucleotide as described herein. In derived from a second alga Such as described herein in com another embodiment, the present invention provides a trans bination with an elongase as described herein produces better genic plant, a transgenic Seed, or a transgenic alga trans results in comparison to fungal or wild-type genes. In another formed by any combination of polynucleotides as described embodiment, transforming a first alga with a combination of herein. In another embodiment, the present invention pro algal genes derived from a second alga, a third alga, etc., Such vides that the transgenic plant is a true-breeding for the poly as described herein produces better results (such as ARA nucleotide?s as described herein. In another embodiment, the production) in comparison to fungal or wild-type genes. In present invention provides a transgenic seed, produced by a another embodiment, transforming a first alga with a combi transgenic plant transformed by the polynucleotide?s as nation of algal desaturase genes derived from a second alga described herein. In another embodiment, a transgenic plant, such as described herein produces better results (such as ARA a transgenic seed, or a transgenic alga as described herein production) in comparison to fungal or wild-type genes. In produces very long-chain polyunsaturated fatty acid (VLC another embodiment, P incisa is the second alga. In another PUFA). In another embodiment, a transgenic plant, a trans embodiment, P incisa is the source of choice for genes that genic seed, or a transgenic alga as described herein produces are involved in the biosynthesis of VLC-PUFA. In another arachidonic acid. In another embodiment, a transgenic plant embodiment, P incisa is the source of choice for genes that or a transgenic Seed as described herein produces DHA. In are involved in the biosynthesis of ARA, DHA, EPA, DGLA, another embodiment, a transgenic plant, a transgenic seed, or or any combination thereof. In another embodiment, P. a transgenic alga as described herein produces DGLA. incisa-derived genes Such as described herein are more effec 0058. In another embodiment, the present invention pro tive alone or in combination than those of other sources. vides a method of producing very long-chain polyunsaturated 0054. In another embodiment, a DNA sequence as fatty acid (VLC-PUFA) in a plant, a plant cell, or an alga described herein such as but not limited to SEQID NO: 4-6 is comprising the step of transforming a plant, a plant cell, oran used to engineer a transgenic organism. In another embodi alga with a polynucleotide as described herein. In another ment, DNA sequences as described herein Such as but not embodiment, the present invention unexpectedly provides an limited to SEQID NO: 4-6 are used to engineer a transgenic utmost efficient method of producing very long-chain poly organism or transform a cell. In another embodiment, DNA unsaturated fatty acid (VLC-PUFA) in a plant, a plant cell, or sequences as described herein such as but not limited to SEQ an alga comprising the step of transforming a plant, a plant ID NO: 4-6 and 8 are used to engineer a transgenic organism cell, or an alga with a polynucleotide as described herein. In or transform a cell. In another embodiment, the DNA another embodiment, a VLC-PUFA is produced from Y-lino sequences comprise the sequences provided in SEQID NO: lenic acid (GLA). 4-6 and 8 or variants of these sequences due, for example: 0059. In another embodiment, a VLC-PUFA is produced base Substitutions, deletions, and/or additions. from stearidonic acid (SDA). In another embodiment, a VLC 0055. In another embodiment, the present invention pro PUFA is produced from GLA, SDA, or their combination. In vides transgenic plant oils enriched with VLC-PUFA. In another embodiment, a VLC-PUFA comprises 20 carbons. In another embodiment, the present invention provides trans another embodiment, a VLC-PUFA is 20:306 or 20:4c03. In genic alga oils enriched with VLC-PUFA. In another embodi another embodiment, a VLC-PUFA is produced by the pro ment, the present invention provides the reconstitution of tein/s as described herein in a cell or a plant, a plant cell, oran C20-VLC-PUFA biosynthesis in oil-synthesizing seeds of alga. In another embodiment, a VLC-PUFA is produced by higher plants. In another embodiment, the present invention the protein/s as described herein in a cell, a plant, a plant cell, provides expanded use by enhancement of the levels of ARA, or an alga under oleogenic conditions. In another embodi DGLA, DHA, EPA, or a combination thereof in the trans ment, an unexpected amount of VLC-PUFA is produced by genic plants. the protein/s as described herein in a cell, an alga, or a plant 0056. In another embodiment, the present invention pro under nitrogen starvation conditions. vides an expression vector comprising the polynucleotide as 0060. In another embodiment, a cell is a eukaryotic cell. In described herein. In another embodiment, the present inven another embodiment, a cell is a prokaryotic cell. In another tion provides a combination of expression vectors each com embodiment, a cell is a plant cell. In another embodiment, a prising a polynucleotide as described herein. In another cell is an algal cell. In another embodiment, a cell is a trans embodiment, the present invention provides an expression fected cell. In another embodiment, a cell is transiently trans vector comprising a combination of polynucleotides as fected with a polynucleotide or a combination of polynucle described herein. In another embodiment, the present inven otides as described herein. In another embodiment, a cell is tion provides a plant specific expression vector comprising stably transfected with a polynucleotide or a combination of the polynucleotide or combination of polynucleotides as polynucleotides as described herein. In another embodiment, described herein. In another embodiment, the present inven the present invention provides a method of enhancing oil tion provides an algal specific expression vector comprising storage, EPA accumulation, DHA accumulation, ARA accu the polynucleotide or combination of polynucleotides as mulation, DGLA accumulation, or a combination thereof in a described herein. In another embodiment, the present inven cell, comprising the step of transforming a cell with a poly tion provides a cell comprising the expression vector/s as nucleotide as described herein. In another embodiment, the described herein. In another embodiment, the expression vec present invention provides a method of enhancing oil storage, torts is contained within an agrobacterium. In another EPA accumulation, DHA accumulation, ARA accumulation, embodiment, a cell is a plant cell or an algal cell. In another DGLA accumulation, or a combination thereof in a cell, US 2013/0019341 A1 Jan. 17, 2013 comprising the step of transfecting a cell with a polynucle engineered to express a combination of proteins as described otide as described herein. In another embodiment, the present herein. In another embodiment, an engineered organism is invention provides a method of enhancing oil storage, EPA engineered to express elevated levels of the protein or a com accumulation, DHA accumulation, DGLA accumulation, bination of proteins. In another embodiment, an engineered ARA accumulation, or a combination thereof in a cell, com plant as described herein is used for manufacturing desired prising the step of transforming a cell with a combination of PUFAs such as but not limited to ARA. In another embodi polynucleotides as described herein. In another embodiment, ment, an engineered plant as described herein is used for the present invention provides a method of enhancing oil manufacturing desired PUFAs such as ARA at a reduced cost. storage, EPA accumulation, DHA accumulation, ARA accu 0064. In another embodiment, an engineered organism mulation, DGLA accumulation, or a combination thereof in a comprises a synthetic pathway for the production of a protein. cell or a multicellular organism, comprising the step of trans In another embodiment, an engineered organism comprising forming a cell or a multicellular organism with a polynucle a synthetic pathway for the production of the protein allows otide as described herein. In another embodiment, the present greater control over the production of PUFAs by the pathway invention provides a method of enhancing oil storage, EPA by an organism. In another embodiment, the pathway accumulation, DGLA accumulation, DHA accumulation, includes but is not limited to A-fatty acid desaturase, and/or ARA accumulation, or a combination thereof in a multicel A-fatty acid desaturase. lular organism, comprising the step of transforming a multi 0065. In another embodiment, an engineered cell, plant or cellular organism with a combination of polynucleotides as seed comprises an oligonucleotide as described herein. In described herein. In another embodiment, the multicellular another embodiment, an engineered plant or seed produces a organism or cell is grown under nitrogen starvation condi protein as described herein and comprises an oligonucleotide tions, oleogenic conditions, or a combination thereof. as described herein. In another embodiment, an engineered 0061. In another embodiment, transformation as used plant or seed produces proteins as described herein and com herein comprises “transduction”. In another embodiment, prises oligonucleotides as described herein. transformation as used herein comprises transfection. In 0066. In another embodiment, the invention provides a another embodiment, transformation as used herein com method of producing very long-chain polyunsaturated fatty prises "conjugation'. In another embodiment, transformation acid (VLC-PUFA) in a cell, a plant, a plant cell, or an alga, as used herein applies to eukaryotic and prokaryotic cells. In comprising the step of transforming a plant, a plant cell, oran another embodiment, transformation as used herein com alga with a polynucleotide as described herein, thereby pro prises the insertion of new genetic material into nonbacterial ducing a VLC-PUFA in a plant, a plant cell, or an alga. In cells including animal and plant cells. another embodiment, the invention provides a method of 0062. In another embodiment, the present invention pro producing very long-chain polyunsaturated fatty acid (VLC vides a method of enhancing oil storage, EPA accumulation, PUFA) in a cell, a plant, a plant cell, oran alga, comprising the DHA accumulation, DGLA accumulation, ARA accumula step of transforming a plant, a plant cell, or an alga with an tion, or a combination thereof in a plant cell, comprising the exogenous polynucleotide as described herein, thereby pro step of transforming a plant cell with a polynucleotide as ducing a VLC-PUFA in a cell, a plant, a plant cell, or an alga. described herein. In another embodiment, the present inven In another embodiment, the invention provides a method of tion provides a method of enhancing oil storage, EPA accu producing very long-chain polyunsaturated fatty acid (VLC mulation, DHA accumulation, ARA accumulation, DGLA PUFA) in a cell, a plant, a plant cell, oran alga, comprising the accumulation, or a combination thereof in a plant cell, com step of transforming a plant, a plant cell, or an alga with a prising the step of transforming a plant cell with a combina vector comprising an exogenous polynucleotide as described tion of polynucleotides as described herein. In another herein, thereby producing a VLC-PUFA in a cell; a plant, a embodiment, the present invention provides a method of plant cell, or an alga. In another embodiment, the invention enhancing oil storage, EPA accumulation, DHA accumula provides a method of producing very long-chain polyunsatu tion, ARA accumulation, DGLA accumulation, or a combi rated fatty acid (VLC-PUFA) in a cell, a plant, a plant cell, or nation thereof in a plant, comprising the step of transforming an alga, comprising the step of transforming a plant, a plant a plant with a polynucleotide as described herein. In another cell, or an alga with a combination of vectors comprising a embodiment, the present invention provides a method of combination of exogenous polynucleotides as described enhancing oil storage, EPA accumulation, DHA accumula herein, thereby producing a VLC-PUFA in a cell, a plant, a tion, ARA accumulation, DGLA accumulation, or a combi plant cell, or an alga. In another embodiment, the invention nation thereof in a plant, comprising the step of transforming provides a method of producing very long-chain polyunsatu a plant with a combination of polynucleotides as described rated fatty acid (VLC-PUFA) in a cell, a plant, a plant cell, or herein. In another embodiment, the plant or plant cell is an alga, comprising the step of transforming a cell, a plant, a grown under nitrogen starvation conditions, oleogenic con plant cell, or an alga with a combination of exogenous poly ditions, or a combination thereof. nucleotides as described herein, thereby producing a VLC 0063. In another embodiment, the invention further pro PUFA in a cell, a plant, a plant cell, or an alga. vides an engineered organism, Such as a transgenic plant. In 0067. In another embodiment, the invention provides that another embodiment, the invention further provides an engi a plant, a cell, a plant cell, or an alga as described herein is neered organism, Such as a transgenic seed. In another treated or supplemented with linoleic acid (LA; 18:2006), embodiment, the invention further provides an engineered O-linolenic acid (ALA; 18:3(O3), oleic acid (18:1), dihomo organism, such as a transgenic alga. In another embodiment, gamma-linolenic acid (20:3c)6), phosphatidylcholine (PC), the invention further provides an engineered organism, Such diacylglyceroltrimethylhomoserine (DGTS), phosphatidyle as a transgenic animal. In another embodiment, an engineered thanolamine (PE), or any combination thereof, before trans organism is engineered to express a protein as described formation, after transformation, during transformation or a herein. In another embodiment, an engineered organism is combination thereof. US 2013/0019341 A1 Jan. 17, 2013

0068. In another embodiment, the invention provides that for preparing peptidomimetic compounds are well known in the VLC-PUFA is eicosapentaenoic acid (EPA, 20:5c)3). In the art and are specified, for example, in Quantitative Drug another embodiment, the invention provides that the VLC Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Perga PUFA is docosahexaenoic acid (DHA, 22:6c)3). In another mon Press (1992), which is incorporated by reference as if embodiment, the invention provides that the VLC-PUFA is fully set forth herein. Further details in this respect are pro arachidonic acid (ARA, 20:4()6). In another embodiment, the vided hereinunder. invention provides that a cell, a plant, or an alga transformed 0074. In some embodiments, polypeptide bonds ( CO by a polynucleotide or a combination of polynucleotides as NH ) within the polypeptide are substituted. In some described herein, is grown under oleogenic conditions. In embodiments, the polypeptide bonds are substituted by another embodiment, the invention provides that a cell, a N-methylated bonds ( N(CH3)—CO ). In some embodi plant, or an alga transformed by a polynucleotide or a com ments, the polypeptide bonds are substituted by ester bonds bination of polynucleotides as described herein, is grown ( C(R)H C O—O C(R) N ). In some embodi under nitrogen starvation conditions. ments, the polypeptide bonds are substituted by ketomethyl 0069. In another embodiment, the invention provides that ene bonds ( CO—CH2—). In some embodiments, the producing very long-chain polyunsaturated fatty acid (VLC polypeptide bonds are substituted by C.-aza bonds ( NH PUFA) is enhancing oil storage, arachidonic acid accumula N(R)—CO ), wherein R is any alkyl, e.g., methyl, carbo tion, eicosapentaenoic acid accumulation, docosahexaenoic bonds (—CH2—NH ). In some embodiments, the polypep acid accumulation, or a combination thereof that a cell, a tide bonds are substituted by hydroxyethylene bonds ( CH plant, or an alga transformed by a polynucleotide or a com (OH)—CH2—). In some embodiments, the polypeptide bination of polynucleotides as described herein bonds are substituted by thioamide bonds (—CS NH ). In 0070. In another embodiment, a PUFA is di-homo some embodiments, the polypeptide bonds are substituted by gamma-linolenic acid, arachidonic acid, eicosapentaenoic olefimic double bonds ( CH=CH-). In some embodiments, acid, docosatrienoic acid, docosatetraenoic acid, docosapen the polypeptide bonds are substituted by retro amide bonds taenoic acid or docosahexaenoic acid. In another embodi ( NH CO ). In some embodiments, the polypeptide ment, a PUFA is a 24 carbon fatty acid with at least 4 double bonds are substituted by polypeptide derivatives ( N(R)— bonds. CH2—CO ), wherein R is the “normal side chain, natu 0071. In another embodiment, expression of the protein/s rally presented on the carbon atom. In some embodiments, of the invention in plants or seed requires Subcloning an these modifications occur at any of the bonds along the ORF/s sequence encoding the protein/s into a plant expres polypeptide chain and even at several (2-3 bonds) at the same sion vector, which may comprise a viral 35S promoter, and a time. Nos terminator. In another embodiment, a cassette or pro 0075. In some embodiments, natural aromatic amino acids moter/coding sequence/terminator is then be subcloned into of the polypeptide such as Trp, Tyrand Phe, be substituted for the plant binary transformation vector, and the resulting plas synthetic non-natural acid Such as Phenylglycine, TIC, naph mid introduced into Agrobacterium. In another embodiment, thylelanine (Nol), ring-methylated derivatives of Phe, halo the Agrobacterium Strain transforms the plant. In another genated derivatives of Phe oro-methyl-Tyr. In some embodi embodiment, the. Agrobacterium strain transforms the plant ments, the polypeptides of the present invention include one by the vacuum-infiltration of inflorescences, and the seeds or more modified amino acid or one or more non-amino acid harvested and plated onto selective media containing an anti monomers (e.g., fatty acid, complex carbohydrates, etc.). biotic. In another embodiment, the plasmid confers resistance 0076. In one embodiment, “amino acid' or “amino acids’ to an antibiotic, thus only transformed plant material will is understood to include the 20 naturally occurring amino grow in the presence of an antibiotic. In another embodiment, acid; those amino acid often modified post-translationally in resistant lines are identified and self-fertilized to produce Vivo, including, for example, hydroxyproline, phosphoserine homozygous material. In another embodiment, leaf material and phosphothreonine; and other unusual amino acid includ is analyzed for expression of the protein comprising desatu ing, but not limited to, 2-aminoadipic acid, hydroxylysine, rase activity. In another embodiment, leaf material is ana isodesmosine, nor-valine, nor-leucine and ornithine. In one lyzed for expression of a combination of protein comprising embodiment, “amino acid' includes both D- and L-amino desaturase and elongase activities. acid. 0072. In some embodiments, the terms “protein’.“desatu 0077. In some embodiments, the polypeptides or proteins rase', or “polypeptide' are used interchangeably. In some of the present invention are utilized in a soluble form. In some embodiments, “protein”, “desaturase', or “polypeptide' as embodiments, the polypeptides or proteins of the present used herein encompasses native polypeptides (either degra invention include one or more non-natural or natural polar dation products, synthetically synthesized polypeptides or amino acid, including but not limited to serine and threonine recombinant polypeptides) and peptidomimetics (typically, which are capable of increasing polypeptide or protein solu synthetically synthesized polypeptides), as well as peptoids bility due to their hydroxyl-containing side chain. and semipeptoids which are polypeptide analogs, which 0078. In some embodiments, the polypeptides or proteins have, in Some embodiments, modifications rendering the of the present invention are utilized in a linear form, although polypeptides/proteins even more stable while in a body or it will be appreciated by one skilled in the art that in cases more capable of penetrating into cells. where cyclization does not severely interfere with polypep 0073. In some embodiments, modifications include, but tide characteristics, cyclic forms of the polypeptide can also are not limited to N terminus modification, C terminus modi be utilized. fication, polypeptide bond modification, including, but not 0079. In some embodiments, the polypeptides or proteins limited to, CH2 NH, CH2 S, CH2 S-O, O–C NH, of present invention are biochemically synthesized such as by CH2 O, CH2 CH2, S.–C NH, CH-CH or CF CH, using standard Solid phase techniques. In some embodiments, backbone modifications, and residue modification. Methods these biochemical methods include exclusive solid phase Syn US 2013/0019341 A1 Jan. 17, 2013 thesis, partial Solid phase synthesis, fragment condensation, I0086. In one embodiment, “composite polynucleotide or classical solution synthesis. In some embodiments, these sequence” refers to a sequence, which is at least partially methods are used when the polypeptide is relatively short complementary and at least partially genomic. In one (about 5-15 kDa) and/or when it cannot be produced by embodiment, a composite sequence can include some exonal recombinant techniques (i.e., not encoded by a nucleic acid sequences required to encode the polypeptide of the present sequence) and therefore involves different chemistry. invention, as well as some intronic sequences interposing 0080. In some embodiments, solid phase polypeptide or there between. In one embodiment, the intronic sequences protein synthesis procedures are well known to one skilled in can be of any source, including of other genes, and typically the art and further described by John Morrow Stewart and will include conserved splicing signal sequences. In one Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd embodiment, intronic sequences include cis acting expres Ed., Pierce Chemical Company, 1984). In some embodi sion regulatory elements. ments, synthetic polypeptides or proteins are purified by pre I0087. In one embodiment, the polynucleotides of the parative high performance liquid chromatography Creighton present invention further comprise a signal sequence encod T. (1983) Proteins, structures and molecular principles. WH ing a signal peptide for the secretion of the polypeptides of the Freeman and Co. N.Y., and the composition of which can be present invention. In one embodiment, following expression, confirmed via amino acid sequencing by methods known to the signal peptides are cleaved from the precursor proteins one skilled in the art. resulting in the mature proteins. 0081. In some embodiments, recombinant protein tech I0088. In some embodiments, polynucleotides of the niques are used to generate the polypeptides of the present present invention are prepared using PCR techniques or any invention. In some embodiments, recombinant protein tech other method or procedure known to one skilled in the art. In niques are used for generation of relatively long polypeptides Some embodiments, the procedure involves the legation of (e.g., longer than 18-25 amino acid). In some embodiments, two different DNA sequences (See, for example, “Current recombinant protein techniques are used for the generation of Protocols in Molecular Biology', eds. Ausubel et al., John large amounts of the polypeptide of the present invention. In Wiley & Sons, 1992). Some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, I0089. In one embodiment, polynucleotides of the present Studieretal. (1990) Methods in Enzymol. 185:60-89, Brisson invention are inserted into expression vectors (i.e., a nucleic et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) acid construct) to enable expression of the recombinant EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO.J. 3:1671 polypeptide. In one embodiment, the expression vector of the 1680 and Broglietal, (1984) Science 224:838-843, Gurley et present invention includes additional sequences which render al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & this vector Suitable for replication and integration in prokary Weissbach, 1988, Methods for Plant Molecular Biology, Aca otes. In one embodiment, the expression vector of the present demic Press, NY. Section VIII, pp. 421-463. invention includes additional sequences which render this 0082 In one embodiment, a polypeptide or protein of the vector Suitable for replication and integration in eukaryotes. present invention is synthesized using a polynucleotide In one embodiment, the expression vector of the present encoding a polypeptide or protein of the present invention. In invention includes a shuttle vector which renders this vector Some embodiments, the polynucleotide encoding a polypep Suitable for replication and integration in both prokaryotes tide of the present invention is ligated into an expression and eukaryotes. In some embodiments, cloning vectors com vector, comprising a transcriptional control of a cis-regula prise transcription and translation initiation sequences (e.g., tory sequence (e.g., promoter sequence). In some embodi promoters, enhancers) and transcription and translation ter ments, the cis-regulatory sequence is suitable for directing minators (e.g., polyadenylation signals). constitutive expression of the polypeptide of the present 0090. In one embodiment, a variety of prokaryotic or invention. In some embodiments, the cis-regulatory sequence eukaryotic cells can be used as host-expression systems to is suitable for directing tissue specific expression of the express the polypeptides of the present invention. In some polypeptide of the present invention. In some embodiments, embodiments, these include, but are not limited to, microor the cis-regulatory sequence is suitable for directing inducible ganisms, such as bacteria transformed with a recombinant expression of the polypeptide of the present invention. In bacteriophage DNA, plasmid DNA or cosmid DNA expres another embodiment, a polypeptide is a protein comprising a sion vector containing the polypeptide coding sequence; desaturase as described herein. yeast transformed with recombinant yeast expression vectors 0083. In another embodiment, the polynucleotide com containing the polypeptide coding sequence; plant cell sys prises a genomic polynucleotide sequence. In another tems infected with recombinant virus expression vectors embodiment, the polynucleotide comprises a composite (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, polynucleotide sequence. TMV) or transformed with recombinant plasmid expression 0084. In one embodiment, the phrase “a polynucleotide' vectors, such as Tiplasmid, containing the polypeptide cod refers to a single or double stranded nucleic acid sequence ing sequence. which be isolated and provided in the form of an RNA 0091. In some embodiments, non-bacterial expression sequence, a complementary polynucleotide sequence systems are used (e.g., plant expression systems) to express (cDNA), a genomic polynucleotide sequence and/or a com the polypeptide of the present invention. posite polynucleotide sequences (e.g., a combination of the 0092. In one embodiment, yeast expression systems are above). used. In one embodiment, algae expression systems are used. 0085. In one embodiment, “genomic polynucleotide In one embodiment, plant expression systems are used. In one sequence” refers to a sequence derived (isolated) from a chro embodiment, a number of vectors containing constitutive or mosome and thus it represents a contiguous portion of a inducible promoters can be used in yeast as disclosed in U.S. chromosome. Pat. No. 5,932,447 which is hereby incorporated in its entirety US 2013/0019341 A1 Jan. 17, 2013

by reference. In another embodiment, vectors which promote ments, may be expressed either by itself or with other genes integration of foreign DNA sequences into the yeast chromo Such as but not limited to elongase, in order to produce cells, Some are used. tissues, algae, and/or plant parts containing higher propor 0093. In another embodiment, expression in a host cell can tions of desired PUFAs or in which the PUFA composition be accomplished in a transient or a stable fashion. In another more closely resembles that of human breast milk. In another embodiment, a host cell is a cell as described herein. In embodiment, the termination region is derived from the 3' another embodiment, transient expression is from introduced region of the gene from which the initiation region was constructs which contain expression signals functional in the obtained or from a different gene. In another embodiment, the host cell, but which constructs do not replicate and rarely termination region usually is selected as a matter of conve integrate in the host cell, or where the host cell is not prolif nience rather than because of any particular property. erating. In another embodiment, transient expression also can 0096. In another embodiment, a plant or plant tissue is be accomplished by inducing the activity of a regulatable utilized as a host or host cell, respectively, for expression of promoter operably linked to the gene of interest. the protein of the invention which may, in turn, be utilized in 0094. In another embodiment, stable expression is the production of polyunsaturated fatty acids. In another achieved by introduction of a construct that integrates into the embodiment, desired PUFAS are expressed in seed. In host genome. In another embodiment, stable expression com another embodiment, methods of isolating seed oils are prises autonomously replication within the host cell. In known in the art. In another embodiment, seed oil compo another embodiment, stable expression of the polynucleotide nents are manipulated through the expression of the protein of of the invention is selected for through the use of a selectable the invention in order to provide seed oils that can be added to marker located on or transfected with the expression con nutritional compositions, pharmaceutical compositions, ani struct, followed by selection for cells expressing the marker. mal feeds and cosmetics. In another embodiment, a vector In another embodiment, stable expression results from inte which comprises a DNA sequence encoding the protein as gration, the site of the constructs integration can occur ran described herein is linked to a promoter, and is introduced domly within the host genome or can be targeted through the into the plant tissue or plant for a time and under conditions use constructs containing regions of homology with the host sufficient for expression of the protein. genome Sufficient to target recombination with the host locus. 0097. In another embodiment, a vector as described herein In another embodiment, constructs are targeted to an endog comprises additional genes that encode other enzymes, for enous locus, all or some of the transcriptional and transla example, elongase, A4-desaturase, a different A5-desaturase, tional regulatory regions can be provided by the endogenous a different A6-desaturase. A 10-desaturase, a different A12 locus. desaturase, A15-desaturase. A 17-desaturase. A 19-desaturase, 0095. In another embodiment, an expression of a protein or any combination thereof. In another embodiment, the plant as described herein comprising desaturase activity includes tissue or plant produces the relevant substrate upon which the functional transcriptional and translational initiation and ter enzymes act or a vector encoding enzymes which produce mination regions that are operably, linked to the DNA encod Such Substrates may be introduced into the plant tissue, plant ing the protein comprising a desaturase activity. In another cellor plant. In another embodiment, a Substrate is sprayed on embodiment, an expression of proteins as described herein plant tissues expressing the appropriate enzymes. In another comprising various desaturase activities includes functional embodiment, the invention is directed to a transgenic plant transcriptional and translational initiation and termination comprising the above-described vector, wherein expression regions that are operably linked to the DNA encoding the of the nucleotide sequence of the vector results in production proteins comprising desaturase activity. In another embodi of a polyunsaturated fatty acid in, for example, the seeds of ment, an expression of proteins as described herein compris the transgenic plant. ing desaturase and elongase activities includes functional 0098. In another embodiment, the regeneration, develop transcriptional and translational initiation and termination ment, and cultivation of plants from single plant protoplast regions that are operably linked to the DNA encoding each transformants or from various transformed explants is well protein comprising a desaturase or elongase activity. In known in the art (for example: Weissbach and Weissbach, In: another embodiment, transcriptional and translational initia Methods for Plant Molecular Biology, (Eds.), tion and termination regions are derived from a variety of (0099 Academic Press, Inc. San Diego, Calif., (1988)). In nonexclusive sources, including the DNA to be expressed, another embodiment, regeneration and growth process com genes known or Suspected to be capable of expression in the prises the steps of selection of transformed cells, culturing desired system, expression vectors, chemical synthesis, or those individualized cells through the usual stages of embry from an endogenous locus in a host cell. hi another embodi onic development through the rooted plantlet stage. In ment, expression in a plant tissue and/or plant part presents another embodiment, transgenic embryos and seeds are simi certain efficiencies, particularly where the tissue or partis one larly regenerated. In another embodiment, resulting trans which is harvested early, Such as seed, leaves, fruits, flowers, genic rooted shoots are thereafter planted in an appropriate roots, etc. In another embodiment, expression can be targeted plant growth medium such as soil. In another embodiment, to that location in a plant by utilizing specific regulatory regeneration and growth process of algae are known to one of sequences that are lcnown to one of skill in the art. In another skill in the art. In another embodiment, identification, selec embodiment, the expressed protein is an enzyme which pro tion, of transgenic algae are known to one of skill in the art. duces a product which may be incorporated, either directly or 0100. In another embodiment, development or regenera upon further modifications, into a fluid fraction from the host tion of plants containing an exogenous polynucleotide as plant. In another embodiment, expression of a protein of the described herein encodes a protein as described herein and is invention, or antisense thereof, alters the levels of specific well known in the art. In another embodiment, development PUFAs, or derivatives thereof, found in plant parts and/or or regeneration of algae containing an exogenous polynucle plant tissues. The desaturase coding region, in Some embodi otide as described herein encodes a protein as described US 2013/0019341 A1 Jan. 17, 2013

herein and is well known in the art. In another embodiment, the life cycle of, for example, retrovirus, and is the process by the regenerated plants are self-pollinated to provide homozy which a single infected cell produces many progeny virions gous transgenic plants. In another embodiment, pollen that bud off and infect neighboring cells. In one embodiment, obtained from the regenerated plants is crossed to seed-grown the result is that a large area becomes rapidly infected, most of plants of agronomically important lines. In another embodi which was not initially infected by the original viral particles. ment, pollen from plants of these important lines is used to In one embodiment, viral vectors are produced that are unable pollinate regenerated plants. In another embodiment, a trans to spread laterally. In one embodiment, this characteristic can genic plant of the present invention containing a desired be useful if the desired purpose is to introduce a specified polypeptide is cultivated using methods well known to one gene into only a localized number of targeted cells. skilled in the art. 0106. In one embodiment, various methods can be used to 0101. In another embodiment, a variety of methods can be introduce the expression vector of the present invention into utilized for the regeneration of plants from plant tissue. In cells. Such methods are generally described in Sambrook et another embodiment, the method of regeneration will depend al., Molecular Cloning: A Laboratory Manual, Cold Springs on the starting plant tissue and the particular plant species to Harbor Laboratory, New York (1989, 1992), in Ausubelet al., be regenerated. In another embodiment, methods for trans Current Protocols in Molecular Biology, John Wiley and forming dicots, primarily by use of Agrobacterium tumefa Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene ciens, and obtaining transgenic plants are known in the art Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., McCabe et al., Biol. Technology 6:923 (1988), Christou et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vec Plant Physiol. 87:671-674 (1988)); Cheng et al., Plant Cell tors: A Survey of Molecular Cloning Vectors and Their Uses, Rep. 15:653657 (1996), McKently et al., Plant Cell Rep. Butterworths, Boston Mass. (1988) and Gilboa et al. Bio 14:699-703 (1995)); Grant et al., Plant Cell Rep. 15:254-258, techniques 4 (6): 504-512, 1986 and include, for example, (1995). stable or transient transfection, lipofection, electroporation 0102. In another embodiment, transformation of mono and infection with recombinant viral vectors. In addition, see cotyledons using electroporation, particle bombardment, and U.S. Pat. Nos. 5.464,764 and 5.487,992 for positive-negative Agrobacterium are known. In another embodiment, transfor selection methods. mation and plant regeneration are well established in the art. 0107. In one embodiment, plant expression vectors are In another embodiment, assays for gene expression based on used. In one embodiment, the expression of a polypeptide the transient expression of cloned nucleic acid constructs coding sequence is driven by a number of promoters. In some have been developed by introducing the nucleic acid mol embodiments, viral promoters such as the 35S RNA and 19S ecules into plant cells by polyethylene glycol treatment, elec RNA promoters of CaMV Brisson et al., Nature 310:511 troporation, or particle bombardment (Marcotte et al., Nature 514 (1984), or the coat protein promoter to TMV Takamatsu 335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 et al., EMBO J. 6:307-311 (1987) are used. In another (1989); McCarty et al., Cell 66:895-905 (1991); Hattorietal. embodiment, plant promoters are used Such as, for example, Genes Dev. 6:609-618 (1992); Goffet al., EMBO J. 9:2517 the small subunit of RUBISCO Coruzzi et al., EMBO J. 2522 (1990)). 3:1671-1680 (1984); and Brogliet al., Science 224:838-843 0103) In another embodiment, transient expression sys (1984) or heat shock promoters, e.g., soybean hsp17.5-E or tems are used to functionally dissect the oligonucleotides hsp17.3-B Gurley et al., Mol. Cell. Biol. 6:559-565 (1986). constructs. In another embodiment, practitioners are familiar In one embodiment, constructs are introduced into plant cells with the standard resource materials which describe specific using Tiplasmid, Riplasmid, plant viral vectors, direct DNA conditions and procedures for the construction, manipulation transformation, microinjection, electroporation and other and isolation of macromolecules (e.g., DNA molecules, plas techniques well known to the skilled artisan. See, for mids, etc.), generation of recombinant organisms and the example, Weissbach & Weissbach Methods for Plant screening and isolating of clones, (see for example,: Sam Molecular Biology, Academic Press, NY. Section VIII, pp brook et al., Molecular Cloning: A Laboratory Manual, Cold 421-463(1988). Other expression systems such as insects Spring Harbor Press (1989); Maliga et al., Methods in Plant and mammalian host cell systems, which are well known in Molecular Biology, Cold Spring Harbor Press (1995); Birren the art, can also be used by the present invention. et al., Genome Analysis: Detecting Genes, 1, Cold Spring 0108. It will be appreciated that other than containing the Harbor, N.Y. (1998); Birren et al., Genome Analysis: Analyz necessary elements for the transcription and translation of the ing DNA, 2, Cold Spring Harbor, N.Y. (1998); Plant Molecu inserted coding sequence (encoding the polypeptide or pro lar Biology: A Laboratory Manual, eds. Clark, Springer, N.Y. tein), the expression construct of the present invention can (1997)). also include sequences engineered to optimize stability, pro 0104. In one embodiment, the expression vector of the duction, purification, yield or activity of the expressed present invention can further include additional polynucle polypeptide or protein. otide sequences that allow, for example, the translation of 0109. In some embodiments, transformed cells are cul several proteins from a single mRNA Such as an internal tured under effective conditions, which allow for the expres ribosome entry site (IBES) and sequences for genomic inte sion of high amounts of recombinant polypeptide or protein. gration of the promoter-chimeric polypeptide. In some embodiments, effective culture conditions include, 0105. In some embodiments, expression vectors contain but are not limited to, effective media, bioreactor, tempera ing regulatory elements from eukaryotic viruses such as ret ture, pH and oxygen conditions that permit protein produc roviruses are used by the present invention. In some embodi tion. In one embodiment, an effective medium refers to any ments, recombinant viral vectors are useful for in vivo medium in which a cell is cultured to produce the recombi expression of the polypeptides of the present invention since nant polypeptide or protein of the present invention. In some they offer advantages such as lateral infection and targeting embodiments, a medium typically includes an aqueous solu specificity. In one embodiment, lateral infection is inherent in tion having assimilable carbon, nitrogen and phosphate US 2013/0019341 A1 Jan. 17, 2013

Sources, and appropriate salts, minerals, metals and other unsaturated fatty acid produced by the transgenic cell or nutrients, such as vitamins. In some embodiments, cells of the organism as described herein is administered to a human present invention can be cultured in conventional fermenta Subject. In another embodiment, a very long-chain polyun tion bioreactors, shake flasks, test tubes, microtiter dishes and saturated fatty acid produced by the transgenic cell or organ petri plates. In some embodiments, culturing is carried out at ism as described herein is administered to a baby. In another a temperature, pH and oxygen content appropriate for a embodiment, a very long-chain polyunsaturated fatty acid recombinant cell. In some embodiments, culturing conditions produced by the transgenic cell or organism as described are within the expertise of one of ordinary skill in the art. herein is administered to an infant. In another embodiment, a 0110. In some embodiments, depending on the vector and very long-chain polyunsaturated fatty acid produced by the host system used for production, resultant polypeptides or transgenic cell or organism as described herein is adminis proteins of the present invention either remain within the tered to an animal. In another embodiment, a very long-chain recombinant cell, secreted into the fermentation medium, polyunsaturated fatty acid produced by the transgenic cell or secreted into a space between two cellular membranes, or organism as described herein is administered to a mammal. In retained on the outer surface of a cell or viral membrane. another embodiment, a very long-chain polyunsaturated fatty 0111. In one embodiment, following a predetermined time acid produced by the transgenic cell or organism as described in culture, recovery of the recombinant polypeptide or protein herein is administered to a farm animal, a rodent, a pet, or a is effected. lab animal. 0112. In one embodiment, the phrase “recovering the 0119. In another embodiment, the described pharmaceu recombinant polypeptide or protein’ used herein refers to tical and nutritional compositions are utilized in connection collecting the whole fermentation medium containing the with animals (i.e., domestic or non-domestic), as well as polypeptide or protein and need not imply additional steps of humans, as animals experience many of the same needs and separation or purification. conditions as humans. For example, the oil or acids of the 0113. In one embodiment, polypeptides or proteins of the present invention may be utilized in animal or aquaculture present invention are purified using a variety of standard feed Supplements, animal feed Substitutes, animal vitamins or protein purification techniques, such as, but not limited to, in animal topical ointments. affinity chromatography, ion exchange chromatography, fil I0120 In another embodiment, a very long-chain polyun tration, electrophoresis, hydrophobic interaction chromatog saturated fatty acid produced by a protein or a combination of raphy, gel filtration chromatography, reverse phase chroma proteins of the invention is utilized in an infant formula. In tography, concanavalin A chromatography, another embodiment, a very long-chain polyunsaturated fatty chromatofocusing and differential Solubilization. acid produced by a protein or a combination of proteins of the 0114. In one embodiment, to facilitate recovery, the invention is administered to a Subject having a deficiency in expressed coding sequence can be engineered to encode the very long-chain polyunsaturated fatty acid. In another polypeptide or proteins of the present invention and fused embodiment, a very long-chain polyunsaturated fatty acid is cleavable moiety. In one embodiment, a fusion protein can be a polyunsaturated C20 fatty acid. designed so that the polypeptide or protein can be readily I0121. In another embodiment, the isolated protein com isolated by affinity chromatography; e.g., by immobilization prising desaturase activity is used indirectly or directly in the on a column specific for the cleavable moiety. In one embodi production of polyunsaturated fatty acids. In another embodi ment, a cleavage site is engineered between the polypeptide ment, the isolated protein or a combination of isolated pro or protein and the cleavable moiety and the polypeptide or teins comprising desaturase and/or desaturasefelongase protein can be released from the chromatographic column by activities are used indirectly or directly in the production of treatment with an appropriate enzyme or agent that specifi polyunsaturated fatty acids. In another embodiment, cally cleaves the fusion protein at this site e.g., see Booth et “Directly' is meant to encompass the situation where the al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. enzyme directly desaturates the acid. In another embodiment, Biol. Chem. 265:15854-15859 (1990). the latter of which is utilized in a composition. In another 0115. In one embodiment, the polypeptide or protein of embodiment, “Indirectly is meant to encompass the situa the present invention is retrieved in “substantially pure' form. tion where an acid is converted to another acid (i.e., a pathway 0116. In one embodiment, the phrase “substantially pure' intermediate) by the enzyme and then the latter acid is con refers to a purity that allows for the effective use of the protein Verted to another acid by use of a non-desaturase enzyme. In in the applications described herein. another embodiment, a very long-chain polyunsaturated fatty 0117. In one embodiment, the polypeptide or protein of acid produced either directly or indirectly is added to a nutri the present invention can also be synthesized using in vitro tional composition, pharmaceutical compositions, cosmetics, expression systems. In one embodiment, in vitro synthesis and animal feeds, all of which are encompassed by the present methods are well known in the art and the components of the invention. system are commercially available. I0122. In another embodiment, nutritional compositions 0118. In another embodiment, the invention comprises a include any food or preparation for human or animal con process for making a very long-chain polyunsaturated fatty Sumption including for enteral or parenteral consumption, acid produced by the protein or combination of proteins of the which when taken into the body (a) serve to nourish or build invention in a cell as described herein. In another embodi up tissues or Supply energy and/or (b) maintain, restore or ment, the resulting very long-chain polyunsaturated fatty acid Support adequate nutritional status or metabolic functions. In produced by the transgenic cell or organism as described another embodiment, the nutritional composition of the herein is utilized as a food additive. In another embodiment, present invention comprises at least one oil or acid produced a very long-chain polyunsaturated fatty acid produced by the directly or indirectly by use of the protein of the invention and transgenic cellor organism as described herein is utilized as a may either be in a solid or liquid form. In another embodi Supplement. In another embodiment, a very long-chain poly ment, the composition includes edible macronutrients, Vita US 2013/0019341 A1 Jan. 17, 2013

mins and minerals in amounts desired for a particular use. In ment, an oil or acid produce in accordance with the present another embodiment, the amount of Such ingredients will invention may be add to any of these formulas. vary depending on whether the composition is intended for 0128. In another embodiment, a nutritional formula com use with normal, healthy infants, children or adults having prises macronutrients, vitamins, and minerals, as provided specialized needs Such as those which accompany certain herein, in addition to the PUFAs produced in accordance with metabolic conditions (e.g., metabolic disorders). the present invention. In another embodiment, the presence of 0123. In another embodiment, the macronutrients include additional components helps the individual ingest the mini edible fats, carbohydrates and proteins. In another embodi mum daily requirements of these elements. In another ment, edible fats include but are not limited to coconut oil, soy embodiment, an adult and pediatric nutritional formulascom oil, and mono- and diglycerides. In another embodiment, prises the PUFAs as described herein and zinc, copper, folic carbohydrates include but are not limited to glucose, edible acid and antioxidants, or any combination thereof. In another lactose and hydrolyzed search. In another embodiment, pro embodiment, PUFAs produced in accordance with the teins which are utilized in the nutritional composition of the present invention, or derivatives thereof, are added to a invention include but are not limited to Soy proteins, elec dietary Substitute or Supplement, particularly an infant for trodialysed whey, electrodialysed skim milk, milk whey, or mula, for patients undergoing intravenous feeding or for pre the hydrolysates of these proteins. venting or treating malnutrition or other conditions or disease 0124. In another embodiment, vitamins and minerals are states. In another embodiment, PUFAs produced in accor added to the nutritional compositions of the present invention dance with the present invention are used to alter, the com and include but are not limited to: calcium, phosphorus, position of infant formulas in order to better replicate the potassium, Sodium, chloride, magnesium, manganese, iron, PUFA content of human breast milk or to alter the presence of copper, Zinc, Selenium, iodine, and Vitamins A, E, D, C, and PUFAs normally found in a non-human mammal’s milk. the B complex. Other such vitamins and minerals may also be I0129. In another embodiment, parenteral nutritional com added. positions comprising from about 2 to about 30 weight percent 0.125. In another embodiment, components utilized in the fatty acids calculated as triglycerides are encompassed by the nutritional compositions of the present invention will be of present invention. In another embodiment, other vitamins, semi-purified or purified origin. By semi-purified or purified particularly fat-soluble vitamins such as vitamin A, D, E and is meanta material which has been prepared by purification of L-carnitine are also included. In another embodiment, a pre a natural material or by Synthesis. In another embodiment, servative Such as alpha-tocopherol is added in an amount of nutritional compositions of the present invention include but about 0.05-0.5% by weight. are not limited to infant formulas, dietary Supplements, 0.130. In another embodiment, the present invention dietary Substitutes, and rehydration compositions. In another includes a PUFA produced in accordance with the present embodiment, a nutritional composition of the present inven invention or host cells containing them, used as animal food tion may also be added to food even when Supplementation of Supplements to alter an animal's tissue or milk fatty acid the diet is not required. In another embodiment, a composi composition to one more desirable for human or animal con tion is added to food of any type including but not limited to Sumption. margarines, modified butters, cheeses, milk, yogurt, choco I0131. In one embodiment, the polypeptides or protein of late, candy, Snacks, salad oils, cooking oils, cooking fats, the present invention can be provided to the individual perse. meats, fish and beverages. In one embodiment, the polypeptides or proteins of the 0126. In another embodiment, a nutritional composition is present invention can be provided to the individual as part of an enteral nutritional product. In another embodiment, a a pharmaceutical composition where it is mixed with a phar nutritional composition is an adult or pediatric enteral nutri maceutically acceptable carrier. tional product. In another embodiment, a composition is 0.132. In one embodiment, a “pharmaceutical composi administered to adults or children experiencing stress or hav tion” refers to a preparation of one or more of the active ing specialized needs due to chronic or acute disease states. In ingredients described herein with other chemical components another embodiment, a composition comprises, in addition to Such as physiologically suitable carriers and excipients. The polyunsaturated fatty acids produced in accordance with the purpose of a pharmaceutical composition is to facilitate present invention, macronutrients, vitamins and minerals as administration of a compound to an organism. In one embodi described above. In another embodiment, the macronutrients ment, “active ingredient” refers to the polypeptide or protein may be present in amounts equivalent to those present in sequence of interest. human milk or on an energy basis, i.e., on a per calorie basis. I0133. In one embodiment, the present invention provides 0127. In another embodiment, the present invention combined preparations. In one embodiment, “a combined includes an enteral formula comprising polyunsaturated fatty preparation' defines especially a “kit of parts' in the sense acids produced in accordance with the present invention. In that the combination partners as defined above can be dosed another embodiment, an enteral formula is sterilized and sub independently or by use of different fixed combinations with sequently utilized on a ready-to-feed basis or stored in a distinguished amounts of the combination partners i.e., concentrated liquid or powder. In another embodiment, a simultaneously, concurrently, separately or sequentially. In powder is prepared by spray drying the formula prepared as Some embodiments, the parts of the kit of parts can then, e.g., indicated above, and reconstituting it by rehydrating the con be administered simultaneously or chronologically stag centrate. In another embodiment, the present invention gered, that is at different time points and with equal or differ includes an adult and pediatric nutritional formulas. In ent time intervals for any part of the kit of parts. The ratio of another embodiment, adult and pediatric nutritional formulas the total amounts of the combination partners, in some are known in the art and are commercially available (e.g., embodiments, can be administered in the combined prepara SimilacR), Ensure(R), Jevity(R) and AlimentumR) from Ross tion. In one embodiment, the combined preparation can be Products Division, Abbott Laboratories). In another embodi varied, e.g., in order to cope with the needs of a patient US 2013/0019341 A1 Jan. 17, 2013 subpopulation to be treated or the needs of the single patient Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353–365 which different needs can be due to a particular disease, (1989); Lopez-Berestein, ibid., pp. 317-327; see generally severity of a disease, age, sex, or body weight as can be ibid). readily made by a person skilled in the art. 0142. In some embodiments, the protein as described 0134. In one embodiment, the phrase “physiologically herein is in powder form for constitution with a suitable acceptable carrier refers to a carrier or a diluent that does not vehicle, e.g., Sterile, pyrogen-free water based solution, cause significant irritation to a tissue Such as a plant tissue or before use. In another embodiment, compositions are con a cell Such as a plant cell; and does notabrogate the biological tained in a container with attached atomizing means. activity and properties of the protein or polynucleotide of the 0143. In some embodiments, compositions suitable for invention. An adjuvant is included under these phrases. In one use in context of the present invention include compositions embodiment, one of the ingredients included in the physi wherein the proteins or oligonucleotides are contained in an ologically acceptable carrier can be for example polyethylene amount effective to achieve the intended purpose. In one glycol (PEG), a biocompatible polymer with a wide range of embodiment, determination of the effective amount is well solubility in both organic and aqueous media (Mutter et al. within the capability of those skilled in the art. (1979). 0144. Some examples of substances which can serve as 0135) In one embodiment, “excipient” refers to an inert carriers or components thereof are Sugars, such as lactose, substance added to the composition to further facilitate glucose and Sucrose; starches, such as com starch and potato administration of an active ingredient. In one embodiment, starch; cellulose and its derivatives, such as sodium car excipients include calcium carbonate, calcium phosphate, boxymethyl cellulose, ethyl cellulose, and methyl cellulose; various Sugars and types of starch, cellulose derivatives, gela powdered tragacanth; malt, gelatin; talc, Solid lubricants, tin, vegetable oils and polyethylene glycols. Such as Stearic acid and magnesium Stearate; calcium sulfate; 0.136 Techniques for formulation and administration of Vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, peptide to plants or in-vitro are knownto one of skill in the art. olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, Sorbitol, mannitol, and polyeth 0.137 In one embodiment, compositions of the present ylene glycol; alginic acid; emulsifiers, such as the Tween invention are manufactured by processes well known in the brand emulsifiers; wetting agents, such sodium lauryl Sulfate; art, e.g., by means of conventional mixing, dissolving, or coloring agents; flavoring agents; tableting agents, stabiliz lyophilizing processes. ers; antioxidants; preservatives; pyrogen-free water, isotonic 0.138. In one embodiment, compositions for use in accor saline; and phosphate buffer solutions. The choice of a phar dance with the present invention is formulated in conven maceutically-acceptable carrier to be used in conjunction tional manner using one or more physiologically acceptable with the compound is basically determined by the way the carriers comprising excipients and auxiliaries, which facili compound is to be administered. If the Subject compound is to tate processing of the proteins/polynucleotides into prepara be injected, in one embodiment, the pharmaceutically-ac tions. In one embodiment, formulation is dependent upon the ceptable carrier is sterile, physiological saline, with a blood method of administration chosen. compatible Suspending agent, the pH of which has been 0.139. The compositions also comprise, in some embodi adjusted to about 7.4. ments, preservatives. Such as benzalkonium chloride and 0145. In addition, the compositions further comprise bind thimerosal and the like; chelating agents, such as edetate ers (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellu Sodium and others; buffers such as phosphate, citrate and lose, guar gum, hydroxypropyl cellulose, hydroxypropyl acetate; tonicity agents such as Sodium chloride, potassium methyl cellulose, povidone), disintegrating agents (e.g., corn chloride, glycerin, mannitol and others; antioxidants such as starch, potato starch, alginic acid, silicon dioxide, croscar ascorbic acid, acetylcystine, sodium metabisulfote and oth melose Sodium, crospovidone, guar gum, sodium starch gly ers; aromatic agents; Viscosity adjustors, such as polymers, colate), buffers (e.g., Tris-HCl, acetate, phosphate) of various including cellulose and derivatives thereof; and polyvinyl pH and ionic strength, additives such as albuminor gelatin to alcohol and acid and bases to adjust the pH of these aqueous prevent absorption to Surfaces, detergents (e.g., Tween 20, compositions as needed. The compositions also comprise, in Tween 80, Pluronic F68, bile acid salts), protease inhibitors, Some embodiments, local anesthetics or other actives. The Surfactants (e.g., Sodium lauryl Sulfate), permeation enhanc compositions can be used as sprays, mists, drops, and the like. ers, Solubilizing agents (e.g., glycerol, polyethylene glyc 0140. Additionally, suspensions of the active ingredients, erol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, in some embodiments, are prepared as appropriate oily or butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl water based suspensions. Suitable lipophilic solvents or cellulose, hyroxypropylmethyl cellulose), Viscosity increas vehicles include, in some embodiments, fatty oils such as ing agents(e.g., carbomer, colloidal silicon dioxide, ethyl cel sesame oil, or synthetic fatty acid esters such as ethyl oleate, lulose, guar gum), Sweeteners (e.g., aspartame, citric acid). triglycerides or liposomes. Aqueous injection Suspensions preservatives (e.g., Thimerosal, benzyl alcohol, parabens), contain, in some embodiments, Substances, which increase lubricants (e.g., Stearic acid, magnesium Stearate, polyethyl the Viscosity of the Suspension, Such as Sodium carboxym ene glycol, Sodium lauryl Sulfate), flow-aids (e.g., colloidal ethyl cellulose, sorbitol or dextran. In another embodiment, silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl the Suspension also contains Suitable stabilizers or agents, citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, which increase the solubility of the active ingredients to allow Sodium lauryl Sulfate), polymer coatings (e.g., poloxamers or for the preparation of highly concentrated Solutions. poloxamines), coating and film forming agents (e.g., ethyl 0141. In another embodiment, the proteins as described cellulose, acrylates, polymethacrylates) and/or adjuvants. herein can be delivered in a vesicle, in particular a liposome 0146 The compositions also include incorporation of the (see Langer, Science 249:1527-1533 (1990); Treat et al., in proteins or oligonucleotides of the invention into or onto Liposomes in the Therapy of Infectious Disease and Cancer, particulate preparations of polymeric compounds such as US 2013/0019341 A1 Jan. 17, 2013

polylactic acid, polglycolic acid, hydrogels, etc., or onto lipo literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839, Somes, microemulsions, micelles, unilamellar or multilamel 1533,850,752; 3,850,578; 3,853.987; 3,867,517; 3,879,262: lar vesicles, erythrocyte ghosts, or spheroplasts.) Such com 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; positions will influence the physical state, solubility, stability, 4,098,876; 4,879,219; 5,011,771 and 5,281.521: “Oligo rate of in vivo release, and rate of in vivo clearance. nucleotide Synthesis” Gait, M.J., ed. (1984); "Nucleic Acid 0147 Also comprehended by the invention are particulate Hybridization’ Hames, B. D., and Higgins S.J., eds. (1985); compositions coated with polymers (e.g. poloxamers or “Transcription and Translation’ Hames, B.D., and Higgins S. poloxamines) and the proteins or oligonucleotides of the J., eds. (1984): “Animal Cell Culture’ Freshney, R. I., ed. invention coupled to antibodies directed against tissue-spe (1986): “Immobilized Cells and Enzymes' IRL Press, cific receptors, ligands or antigens or coupled to ligands of (1986): “A Practical Guide to Molecular Cloning Perbal, B., tissue-specific receptors. (1984) and “Methods in Enzymology” Vol. 1-317, Academic 0148. In some embodiments, the proteins or oligonucle Press: “PCR Protocols: A Guide To Methods And Applica otides of the invention modified by the covalent attachment of tions”. Academic Press, San Diego, Calif. (1990); Marshaket water-soluble polymers such as polyethylene glycol, copoly al., “Strategies for Protein Purification and Characteriza mers of polyethylene, glycol and polypropylene glycol, car tion—A Laboratory Course Manual CSHL Press (1996); all boxymethyl cellulose, dextran, polyvinyl alcohol, polyvi of which are incorporated by reference. Other general refer nylpyrrolidone or polyproline. In another embodiment, the ences are provided throughout this document. modified proteins or oligonucleotides of the invention exhibit substantially longer half-lives in blood following intravenous Experimental Procedures injection than do the corresponding unmodified compounds. In one embodiment, modifications also increase the proteins or oligonucleotides solubility in aqueous Solution, eliminate Strains And Growth Conditions aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and 0151. Axenic cultures of P incisa were cultivated on reactivity of the compound. In another embodiment, the BG-11 nutrient medium in 250 ml Erlenmeyer glass flasks in desired in vivo biological activity is achieved by the admin an incubator shaker at controlled temperature (25° C.) and istration of Such polymer-compound abducts less frequently illumination (115umol quanta m° S) under an air/CO. or in lower doses than with the unmodified compound. atmosphere (99:1, v/v) and a speed of 170 rpm. For N-star 0149 Additional objects, advantages, and novel features vation experiments, cells of daily-diluted cultures were col of the present invention will become apparent to one ordi lected by centrifugation, washed three times in sterile DDW narily skilled in the art upon examination of the following and resuspended in N-free BG 11 medium. To prepare N-free examples, which are not intended to be limiting. Additionally, BG-11 medium, sodium nitrate was omitted and ferricammo each of the various embodiments and aspects of the present nium citrate was substituted with ferric citrate. Biomass was invention as delineated hereinabove and as claimed in the sampled at time 0, and in 1.5, 3, 7 and 14 days from the onset claims section below finds experimental Support in the fol of N-starvation for determination of growth parameters, and lowing examples. was further used for fatty acid analysis and RNA isolation. Duplicate samples were collected from 3 separate flasks for EXAMPLES each time point and measurement. 0150 Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention Growth Parameters include molecular, biochemical, microbiological and recom binant DNA techniques. Such techniques are thoroughly 0152 Dry weight and chlorophyll contents were deter explained in the literature. See, for example, “Molecular mined as previously described in A. E. Solovchenko, I. Cloning: A laboratory Manual Sambrook et al., (1989); Khozin-Goldberg, Z. Cohen, M. N. Merzlyak, Carotenoid “Current Protocols in Molecular Biology” Volumes I-III to-chlorophyll ratio as a proxy for assay of total fatty acids Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols and arachidonic acid content in the green micro-alga, Pari in Molecular Biology”, John Wiley and Sons, Baltimore, etochloris incisa, J. Appl. Phycol. (2008) 361-366. Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et RNA Isolation al., “Recombinant DNA, Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory 0153 Aliquots of the cultures were filtered through a glass Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory fiber filter (GF-52, Schleicher & Schuell, Germany); cells Press, New York (1998); methodologies as set forth in U.S. were collected by scraping and immediately flash-frozen in Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and liquid nitrogen and stored at -80° C. for further use. Total 5,272,057: “Cell Biology: A Laboratory Handbook'', Vol RNA was isolated by the procedure described by Bekesiova et umes I-III Cellis, J. E., ed. (1994): “Culture of Animal al. (I. Bekesiova, J. P. Nap, L. Mlynarova, Isolation of high Cells—A Manual of Basic Technique' by Freshney, Wiley quality DNA and RNA from leaves of the carnivorous plant Liss, N.Y. (1994), Third Edition: “Current Protocols in Drosera rotundifolia, Plant. Mol. Biol. Rep. 17 (1999) 269 Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites 277), with minor modifications. Three independent RNA iso et al. (eds), “Basic and Clinical Immunology” (8th Edition), lations were conducted for each time point. The total RNA Appleton & Lange, Norwalk, Conn. (1994); Mishell and samples were treated with RNAase-free Baseline-ZEROTM Shigi (eds), “Selected Methods in Cellular Immunology”. W. DNAase (Epicentre Technologies, Madison, Wis., USA) H. Freeman and Co., New York (1980); available immunoas before being used in cDNA synthesis for real-time PCR says are extensively described in the patent and Scientific experiments. US 2013/0019341 A1 Jan. 17, 2013 18

Gene Cloning and ligation to the pYES2 vector (Invitrogen, Carlsbad, 0154 Partial sequences of the A12, A6, A5 desaturase and Calif., USA), the constructs were used to transform S. cerevi actin genes were obtained by PCR (ReddyMix PCR Master siae strain W303 by the PEG/lithium acetate method R. D. Mix, Thermo Scientific, Surrey, UK) using the degenerated Gietz, R. A. Woods, Yeast Transformation by the LiAc/SS primers listed in the Table 1. To generate the full-length Carrier DNA/PEG Method, in: W. Xiao (Ed.), Yeast Proto cDNAs, 3- and 5'-rapid amplification of the cDNA ends cols, Second Edition, Vol. 313, Methods in Molecular Biol (RACE) was performed using a BD SmartTM RACE cDNA ogy, Humana Press Inc, Totowa, N.J., 2006, pp. 107-120). The Amplification Kit (BD Biosciences Clontech, Foster City, yeast cells harboring the empty pYES2 vector were used as Calif., USA). Gene specific primers were designed (Table 1) control. Transformants were selected by uracil prototrophy and RACE PCR reactions were conducted using 5' and on yeast synthetic medium (YSM) lacking uracil (Invitrogen, 3'-RACE-Ready cDNAs made from 1 lug total RNA of Carlsbad, Calif., USA). For functional expression, a minimal N-starved cells with 50x BD Advantage 2 polymerase mix selection medium containing 2% (w/v) raffinose was inocu (Clontech Laboratories Inc., Mountain View, Calif., USA). lated with the pYPiDesA12, pYPiDesA6 or pYPiDesA5 The PCR products of the expected sizes were excised, puri transformants and grown at 27°C. for 24 h in a water bath fied from the gel (Nucleo Spin Extract II purification kit, shaker. Five ml of sterile YSM, containing 1% (w/v) Tergitol Machery-Nagel, Duren, Germany) and ligated into a pCEM 40 and 250 uM of the appropriate fatty acid substrate was T-Easy vector (Promega, Madison, Wis., USA). The full inoculated with raffinose-grown cultures to obtain an OD of length cDNAs were assembled based on the sequences of the 0.2 at 600 nm. Expression was induced by adding galactose to 5' and 3' RACE fragments. a final concentration of 2% (w/v) and cultures were further TABLE 1. Primers used for obtaining partial, 5' and 3' end fragments of actin, A12, A6 and A5 desaturase genes of P. incisa followed by full-length assembly SEQ ID Gene Forward/Reverse primer (Sequence 5' to 3') 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 TCT TOT 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

Restriction sites */ SEO ID Primers used for full-length cloning and yeast transformations NO: DesA12 TGGAATTCAAAATGGGGAAAGGAGGCTG (FWD) EcoRIA23 CTGTCTAGATCAAGCGCGGAACCACAGG Xba I/24 DesA6 TCGAATTCAAAATGTGCCAGGGACAGG (FWD) EcoRI/25 GGCTCTAGACTAGGCCTCAGCTGCCACG Xbal/26 DesA5 CCAAAGCTTAAAATGATGGCTGTAACAGA (FWD) HindIII/27 GCTCTAGACTATCCCACGGTGGCCA Xbal/28

Expression And Functional Characterization. In the grown at 27°C. for 48 h. Cells were harvested by centrifuga Yeast Saccharomyces Cerevisiae tion, washed twice with 0.1% NaHCO, freeze-dried and used for fatty acid analysis. 0155 The open reading frames (ORFs) encoding for the A2, A6, and A5 desaturases were amplified using Pful Jltra II Generation of 5' And 3' End Fragments of the fusion HS DNA polymerase (Stratagene, La Jolla, Calif., Putative P Incisa PUFA Elongase USA) with the respective primer pairs (Table 1). The forward primers contained a restriction site (underlined) and a yeast 0156 To generate the full-length cDNA of the putative translation consensus (double underlined) followed by ATG. PUFA elongase, 3'-and 5'-rapid amplification of the cDNA The reverse primers contained a restriction site (underlined) ends (RACE) was performed using a BD smartTM RACE and a stop codon (double underlined). Following restriction cDNA Amplification Kit (BD Biosciences Clontech, Foster US 2013/0019341 A1 Jan. 17, 2013 19

City, Calif.) according to the manufacturer's manual. To YpPiELO1-transformants and grown at 27°C. for 24h in a amplify the 5'-end, the reverse gene-specific primers (GSP) water bath shaker. Five ml of sterile YSM, containing 1% 5'-CCCGGCTGCTGCCATGCTTCTGTG (EL5R1) (SEQ (w/v) Tergitol-40 and 250 uM of the appropriate fatty acid ID NO: 29) 5'-TGGGGTAGGGAGAGTAGGCCCAAGT was inoculated with raffinose-grown cultures to obtainan OD (EL5RN) (SEQID NO:30) were designed using the Primer3 of 0.2 at 600 nm. Expression was induced by adding galactose online software (http://frodo.wi.mit.edu). Based on the nucle to a final concentration of 2% (w/v) and cultures were further otide sequence of the obtained 5'-end fragment, two forward grown at 27°C. for 48 h. Cells were harvested by centrifuga GSPs, 5'-GCCTACATGTCCTCTGCCGCCTGCTA tion, washed twice with 0.1% NaHCO, freeze-dried and (EL3R1) (SEQ ID NO: 31) and the nested, 5'-GCGGGA used for fatty acid analysis. CATGGGAGGGCTCATCTATACC (EL3R2) (SEQ ID NO: 32), were constructed to amplify the 3'-end of the target gene. RACE PCR reactions were conducted using 5' and 3'-RACE Primer Design And Validation For PiELO1 Ready cDNAs made from lug total RNA of N-starved cells (Elongase) with 50x BD Advantage 2 polymerase mix (Clontech Labo ratories Inc., Mountain View, Calif.). The PCR products of the 0158 Real-Time Quantitative PCR primer pairs were expected size were excised and purified from the gel (Nu designed for the PiELOJ and the house keeping gene 18S cleoSpin Extract II purification kit, Machery-Nagel, Duren, SSU rRNA using the PrimerQuest tool (http://testidtdna. Germany) and ligated into a pGEMT-Easy vector (Promega, com/Scitools/Applications/Primerquest/). Parameters were Madison, Wis.). The full length cDNA corresponding to the P. incisa putative PUFA elongase (designated PiELO1) was set for a primer length of 19 to 26 bp, primer melting tem assembled from the 5' and 3' RACE fragments and its ORF perature of 60.0-1.0°C., and amplicon length of 90 to 150 was further subcloned into a pYES2 vector (Invitrogen, bases. Primer pairs were validated using seven serial fifty Carlsbad, Calif.). fold dilutions of cDNA samples and standard curves were plotted to test for linearity of the response. The primer pairs Expression And Functional Characterization of and primer concentrations with reaction efficiencies of PiELO1 cDNA (Elongase) In the Yeast 100+10% were chosen for quantitative RT-PCR analysis of Saccharomyces Cerevisiae relative gene expression. The nucleotide sequences and char (O157. The ORF encoding for PiELO1 was amplified using acteristics of primers used for quantitative RT-PCR analysis Pful Jltra II fusion HSDNA polymerase (Stratagene, LaJolla, are presented in Table 2. TABLE 2 Parameters of the primers used in RTOPCR reactions Amplicon PCR Forward primer size efficiency Gene Reverse primer (bp) (%) PiFLO1 AAGCTGTACGAGTTTGTGGATACGCT (SEO ID NO : 35) (FWD) 95 92.3 GGATATGGAAGCGTGGTGGTAGA (SEQ ID NO: 36) (REV)

18S TGAAAGACGAACTTCTGCGAAAGCA (SEO ID NO : 37) (FWD) 12 O 96.8 SSU AGTCGGCATCGTTTATGGTTGAGA (SEO ID NO : 38) (REV) rRNA

Calif.) with the forward primer, 5'-AG Calculation of Gene Transcript Levels GAATTCAAAATGGCATTGACGGCGGCCT 0159. The mean fold changes in gene expression were (PUFAEL5RES1) (SEQID NO: 33), containing a restriction calculated according to the 2^' method using the average site (underlined) and a yeast translation consensus followed of threshold cycle (Ct) values from triplicate cDNA-primer by ATG (double underlined) and the reverse primer 5'-CAT samples. The ACt followed by the AACt was calculated from TCTAGATTACTGCAGCTTTTGCTTGGCTGC the average Ct values of the target and the endogenous genes. (PUFAEL3RES2) (SEQID NO. 34) containing a restriction The transcript abundance of the PiELO1 gene was normal site (underlined) and a stop codon (double underlined). The ized to the endogenous control 18S SSU rRNA gene. The amplified sequence was then restricted with EcoRI and Xbal fold-change in gene expression was calculated using 2^^ to (NEB, Ipswich, Mass.). The expected bands were gel-puri find the expression level of the target gene which was nor fied with NucleoSpin Extract II purification kit (Machery malized to the endogenous gene, relative to the expression of Nagel GmbH, Duren, Germany) and ligated into a EcoRI the target gene at time 0. Xbal cut pYES2 vector, yielding YpPiELO1. Saccharomyces cerevisiae strain W303 was transformed with YpPiELO1 by Fatty Acid Analysis the PEG/lithium acetate method. The yeast cells harboring (0160 Fatty acid methyl esters (FAMES) were obtained by the empty pYES2 vector were used as control. Transformants transmethylation of the freeze-dried P incisa or yeast biom were selected by uracil prototrophy on yeast synthetic ass, with dry methanol containing 2% HSO4 (v/v) and heat medium (YSM) lacking uracil (Invitrogen, Carlsbad, Calif.). ing at 80° C. for 1.5 h while stirring under an argon atmo For functional expression, a minimal selection medium con sphere. Gas chromatographic analysis of FAMES was taining 2% (w/v) raffinose was inoculated with the performed on a Thermo Ultra Gas chromatograph (Thermo US 2013/0019341 A1 Jan. 17, 2013 20

Scientific, Italy) equipped with PTV injector, FID detector Real-Time Quantitative PCR and a fused silica capillary column (30 mx0.32 mm; ZB (0163 Template cDNA for real-time quantitative PCR WAXplus, Phenomenex). FAMEs were identified by (RTOPCR) was synthesized using 1 lug of total RNA in a total co-chromatography with authentic standards (Sigma Chemi volume of 20-ul, using oligo dT primer (Reverse-iT TM 1 cal Co., St. Louis, Mo.; Larodan Fine Chemicals, Malmö, Strand Synthesis Kit, ABgene, Surrey, UK). Each 20-uL Sweden) and FAME of fish oil (Larodan Fine Chemicals). Each sample was analyzed in duplicates of three independent cDNA reaction was then diluted 3-fold with PCR grade water. experiments. The structures of fatty acids were confirmed by Primer Design And Validation For Real-Time GC-MS of their pyrrolidine derivatives W.W. Christie, The Quantitative PCR analysis of fatty acids in: W.W. Christie (Ed.), Lipid analysis (0164 Real-Time Quantitative PCR (RTOPCR) primer Isolation, separation, identification and structural analysis of pairs were designed for the PiDes 12, PiDesG, and PiDes5 lipids, Vol. 15. Third edition, The Oily Press, Bridgewater, genes and the house keeping gene actin, Piact using the England, 2003, pp. 205-225 on HP 5890 equipped with a PrimerQuest tool (http://testidtdna.com/Scitools/Applica mass selective detector (HP5971A) utilizing a HP-5 capillary tions/Primerquest/). Primer pairs were validated as described column and a linear temperature gradient from 120 to 300° C. by Iskandarov et al. U. Iskandarov, I. Khozin-Goldberg, R. Ofir, Z. Cohen, Cloning and Characterization of the A6 Poly Lipid Analysis unsaturated Fatty Acid Elongase from the Green Microalga 0161 The biomass of S. cerevisiae was heated with iso Parietochloris incisa, Lipids 44 (2009) 545-554). The nucle propanol at 80°C. for 10 min and lipids were extracted by the otide sequences of primer pairs and the amplicon sizes are method of Bligh-Dyer (1959) E. G. Bligh, W.J. Dyer, Arapid presented in Table 3. TABLE 3

Primers used in RTQPCR experiments

Forward primer / Amplicon Gene Reverse primer size (bp)

PiDes12 5' - GAAGCACCACCAAGGATGAGGT (SEQ ID NO: 39) (FWD) 112 5'-AGCGAGACGAAGATGACCAGGAA (SEQ BD NO: 40) (REV)

PiDesó 5'-ACTTCCTGCACCACCAGGTCTTC (SEO ID NO: 41) (FWD) 112 5'-TCGTGCTTGCTCTTCCACCAGT (SEQ ID NO: 42) (REV)

PiDes5 5'-TAAGTGCCAGGGCTGTGCTAGA (SEO ID NO : 43) (FWD) 11O 5'- GAACTGACCCTCCTCTGTGTCCT (SEO ID NO. 44) (REV)

PiAct 5'- CGTCCAGCTCCACGATTGAGAAGA (SEO ID NO: 45) (FWD) 154 5'-ATGGAGTTGAAGGCGGTCTCGT (SEO ID NO : 46) (REV)

Example 1 method of total lipid extraction and purification, Can. J. Bio chem. Physiol. 37 (1959) 911-917. Total lipid extract was Isolation And Identification of CDNAS For A12, A6, separated into neutral and polar lipids by silica Bond-Elute And A5 Desaturase Genes of P Incisa cartridges (Varian, Calif.) using 1% of ethanol in chloroform (V/v) and methanol to elute neutral and polar lipids, respec 0.165. The partial sequences of the A12, A6 and A5 desatu tively Z. Cohen, S. Didi, Y. M. Heimer. Overproduction of rase gene homologues were isolated using degenerate oligo Y-linolenic and eicosapentaenoic acids by algae, Plant nucleotides (Table 1) targeting conserved amino acid motifs Physiol. 98 (1992) 569-572). identified in algae, lower plants and fungi. A partial sequence of the actin gene was amplified to be used as a housekeeping 0162 Polar lipids were separated into individual lipids by gene in RTOPCR experiments. two dimensional TLC on Silica Gel 60 glass plates (10x10 (0166 Partial sequences of 503, 558 and 636 bp, corre cm, 0.25 mm thickness (Merck, Darmstadt, Germany) sponding to the A12, A6, and A5 desaturase genes, respec according to Khozin et al. II. Khozin, D. Adlerstein, C. tively, were used for designing gene specific primers that Bigogno, Y. M. Heimer, Z. Cohen, Elucidation of the Biosyn were used to amplify the 5’- and 3'-ends of the expected genes. thesis of Eicosapentaenoic Acid in the Microalga Porphy Assembling the 5' and 3' RACE PCR product sequences ridium cruentum (II. Studies with Radiolabeled Precursors), resulted in the identification of three cDNA clones with Plant Physiol. 114 (1997) 223-230). Neutral lipids were sequence homologies to known A12, A6, and A5 desaturase resolved with petroleum ether:diethyl ether:acetic acid (70: genes. The full-length cDNAS corresponding to A12, A6, and 30:1, V/v/v). Lipids on TLC plates were visualized by brief A5 desaturase genes were thus designated PiDes 12, PiDesG. exposure to iodine vapors, scraped from the plates and were and PiDes5. The ORFs for PiDes 12, PiDesG and PiDes5 transmethylated for the fatty acid analysis as previously genes were 1140, 1443, and 1446 by in length, respectively, described. coding for the corresponding predicted proteins of 380, 481 US 2013/0019341 A1 Jan. 17, 2013 and 482 amino acids. The predicted amino acid sequence of cells harboring the empty vector, pYES2 (control) did not PiDes12 is 64% and 62% identical to that of Chlorella vul demonstrate desaturation activity on the added Substrates garis (BAB78716) and Chlamydomonas reinhardtii (XP (FIG. 3). 001691669), respectively, while it shares more than 50% identity with those of higher plants. It contains three con (0169 PiDesG and PiDes5 expressions were induced in the served histidine motifs HXXXH, HXXHII and HXXHH. The presence of the main (06 substrates for A6 or A5 fatty acid deduced amino acid sequence of PiDesG is 52% and 51% desaturases, 18:2006 and 20:3 (D6, respectively. New peaks identical to those of the A6 desaturases from the liverwort M. corresponding to 18:3()6 and 20:4(06, respectively, were polymorpha (AAT85661) and the moss Ceratodon pur detected, confirming the predicted function of PiDesG and pureus, respectively (CAB94993). It is also 45% identical to PiDes5. The expression of PiDesG in the presence of 18:303 the M. alpina A6 desaturase (ABN69090). PiDes5 shares 55 resulted in the appearance of the corresponding A6 desatura and 51% identity with the A5 desaturase from the microalgae tion product 18:4c)3 (FIG. 3). PiDesG desaturase was neither M. squamata (CAQ30478), and O. tauri (CAL57370), active on endogenous yeast fatty acids nor on external 18:1. respectively, and 54% with that from M. polymorpha PiDesG was not active on 20:36c)3 either, whereas PiDes5 (AAT85663) but is only 36% identical to the M. alpina A5 desaturated it to the non-methylene-interrupted 20:4^'''' desaturase (AAC72755). Both PiDesG and PiDes5 contain 17. PiDes5 converted 20:4c03 into the respective A5 product, N-terminal fused cytochrome b5 domain including the 20:5c)3 (EPA) as well as the added 18:1 into the non-meth HPGG motif and the three histidine boxes found to be con ylene-interrupted 18:2^ (FIG. 3). The A5 position on served in front-end desaturases. The three characteristically 18:2^ was determined by a characteristic peak of m/z-180 conserved histidine-rich motifs with amino acid patterns of on the GC-MS spectra of its pyrrolidine derivative (not HDexxH, HxxHH.QxxHH in A6 desaturases, and HDxxH, shown). The presence of 18:2 was also observed in the QHXXXHH, QXXHH in A5 desaturases are also present in chromatograms of the PiDes5 transformant supplied with PiDesG and PiDes5, respectively (FIG. 1). C20 fatty acids. In addition, a tiny peak, tentatively identified as 18:4'''''' was present on the chromatogram of the Phylogenetic Analysis PiDes5 transformant fed with 18:30 (Table 4). (0167. An unrooted phylogenetic tree (FIG. 2) of PiDes 12, PiDesG. PiDes5 and several functionally characterized TABLE 4 desaturases from all three groups were constructed to identify their functional and phylogenetic relationships by the neigh Conversion percent of the Supplied fatty acids bor-joining program in MEGA4K. Tamura, J. Dudley, M. by PiDesG and PiDess in S. cerevisiae Nei, S. Kumar, MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0, Mol. Biol. Evol. 24 Fatty acid Desaturase product and conversion (%)* (2007) 1596-1599. The deduced amino acid sequence of PiDes12 is closely related to A12 desaturases of green algae Substrate PiDesA6 PiDeSAS and very similar to those of higher plants. The sequences of PiDes6 and PiDes5 cluster with A6 and A5 desaturases, 18:1A9 18:2A-9 (4.2) respectively, from algae, moss and fungi. PiDes6 is highly 18:249. 12 18:2A6.9, 12 (5.1) similar to the M. polymorpha (MpDES6) and P tricornutum 18:3A9, 12, 15 18:4A69. 12, 15 (4.5) 18:4A5,9,12,158 (14) (PtD6p) A6 desaturases, while PiDes5 appears to be closely 18:346, 9, 12 related to the A5 desaturase from the moss M. polymorpha 20:3A11, 14, 17 20:4A5, 11, 14, 17 (10.0) and shares more sequence similarity with the A5 desaturase 20:3A8, 11, 14 20:4A5, 8, 11.14 (164) from the chlorophytes M. squamata and O. tauri than with 20:4A8, 11, 14, 17 20:5A5.8, 11, 14, 17(17.1) those of fungal origin. However, both A6 or A5 desaturases from M. squamata and O. tauri appear to be structurally more *calculated as the ratio of product(substrate + product) similar to each other than to any of the known desaturases tentative identification from either group. 0170 A kinetic analysis of ARA emergence was con Example 2 ducted in total fatty acids of the PiDes5 transfomiant during 24h following the addition of DGLA. Results showed that ARA peak was evident already after 3 h (corresponding to Functional Expression of PiDes 12, PiDesG, And 10.9% substrate conversion) with a gradual but slow increase PiDess In S. Cerevisiae (up to 15.1% conversion) after 24h. Fatty acid analysis of the 0168 The functional activities of the proteins encoded by major polar and neutral lipids of the yeast transformed with PiDes 12, PiDesG and PiDes5 were examined by heterologous pYPiDes5 was performed 24 h after feeding with 20:3c)6 to expression in S. cerevisiae. To this aim, the pYES2 constructs study the pattern of distribution of ARA within individual pYPiDes12, pYPiDesG and pYPiDes5 containing the ORFs lipids. In the transformed yeast, ARA appeared in all major for PiDes 12, PiDesG, and PiDes5, respectively, were trans phospholipids (Table 4), with the highest proportion detected formed into S. cerevisiae. GC analysis of the FAMEs of the in PC. It was also present in the neutral lipids, TAG, free fatty yeast transformed with pYPiDes 12, revealed an appearance acids (FFA), diacylglycerol (DAG) and sterol esters (SE). of a small peak corresponding to 18:2 (0.3% of TFA; not Taking into account that PC, a major phospholipid of S. shown). An attempt to improve the expression of the recom cerevisiae, constituted for about 16% of total lipids (Table 5), binant protein and to increase the activity by the modification it is obvious that PC allocated the main part of ARA attached of yeast translation consensus was not successful. The yeast to phospholipids. US 2013/0019341 A1 Jan. 17, 2013 22

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 % % 20:406 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

Example 3 gene. The assembled complete 867 by cINA sequence, des ignated as PiELO1, preceded and followed by 22 and 150 by Expression Profiles of PiDes12, PiDesG, And PiDes5 nucleotides at 5' and 3' UTR, respectively. PiELO1 contained Under Nitrogen Starvation an ORF of 289 predicted amino acid residues consistent with 0171 To use actin as a house-keeping gene in quantitative functionally characterized PUFA elongase ORFs from fungi, real-time PCR experiments, a partial fragment (503 bp) of the lower plants and algae (FIG. 5). The deduced amino acid P incisa actin gene was amplified using the primers whose sequence of the PiELO1 was 50% identical to O. tauri and M. design was based on the C. reinhardtii actin cDNA (XM polymorpha A6 PUFA elongase, while sharing 48 and 44% 001699016). Indeed, the expression level of the actingene did identity with P. patens A6 elongase and M. polymorpha A5 not significantly change throughout the nitrogen starvation. elongase, respectively. The PiELO1 is also similar, yet with a PiDes 12, PiDesG, and PiDes5 were upregulated following the lower score, to A6 elongases of fungal origin. It shares 40 and transfer to Nitrogen starvation, reaching the highest expres 36% identity with the A6 PUFA elongases of Thraus sion level on day 3 and decreasing thereafter to a level about tochytrium and M. alpina (not included in the alignment), 15 to 20 fold higher than that at time 0 (FIG. 4). Both the respectively. PiDes 12 and PiDes5 genes were expressed at levels approxi 0173 The predicted amino acid sequence of the PiELO1 mately 65 to 70 fold higher on day 3 than at time 0, while the contained four conserved motifs that are characteristic for PiDesG transcript was about 45 fold higher (FIG. 4). The PUFA elongases (FIG. 5, highlighted). The hydropathy plot expression patterns of PiDes 12, PiDesG, and PiDes5 corre of the PiELO1 deduced amino acid sequences was obtained lated with the enhanced biosynthesis of ARA in P incisa cells using the algorithm available in the DAS transmembrane (Table 6). prediction server (http://www.sbc.su.se/~miklos/DAS/). The TABLE 6 Major fatty acid composition of F 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 two strictly hydrophobic transmembrane domains were found about 50 amino acids downstream and upstream from Identification And Characterization of PiFLO1 the N and C termini, respectively, while the two less hydro (0172. The BLASTX analysis (http://www.ncbi.nlm.nih. phobic domains were located about 100 amino acids down gov/blast) of clones obtained through subtractive hybridiza stream and upstream from the N and C termini, respectively tion revealed a clone of 141 bp whose putative amino acid (FIG. 6). sequence was highly homological to the C-terminal region of PUFA elongases. Using GSP primers, the 870 by 5'-end frag Example 2 ment was amplified and the sequence information was used to Phylogenetic Analysis obtain the 3' end fragment from the 3' RACE Ready cDNA. Alignment of the 800 by 3'-end sequence with that of the 0.174. An unrooted phylogenetic tree of the PiELO1 and 5'-end fragment provided an overlapping nucleotide several functionally characterized PUFA elongases was con sequence and included the partial 141 bp sequence, thus structed to identify their functional and phylogenetic relation confirming the amplification of both ends of the expected ships by the neighbor-joining program in MEGA4. Accord US 2013/0019341 A1 Jan. 17, 2013

ing to FIG. 7 one can see that PiELO1 falls into a group of Goldberg, R. Ofir, Z. Cohen, Cloning and Characterization of PUFA elongases of lower eukaryotes. Although the group the A6 Polyunsaturated Fatty Acid Elongase from the Green contains mostly PUFA elongases with A6 activity, some A5 Microalga Parietochloris incisa, Lipids 44 (2009) 545-554. elongases, e.g., that of M. polymorpha and Leishmania infan represent a set of P incisa genes involved in the biosynthesis tum, are more related to A6 elongases of lower eukaryotes of ARA. U. Iskandarov et al., 2009 is incorporated by refer than to A5 elongases of higher eukaryotes. PiELO1 makes a ence as if fully set forth herein. closely related subgroup with OtBLO1, MpBLO1, MpBLO2 0.178 The his-boxes of A12, A6 and A5 desaturases includ and PpELO1, the OtELO1 being the closest one. ing PiDes 12, PiDesG, and PiDes5 are detailed in Table 8.

Functional Expression of PiELO1 In S. Cerevisiae TABLE 8 (0175 To characterize the enzymatic activity of PiELO1, the pYES2 plasmid containing the PiELO1 ORF downstream Conserved histidine rich motifs of A12, A6, and A5 desaturases of the GAL1 promoter was transformed into S. cerevisiae. Des A12 HECxH HxxHH HxxHH The PiELO1 was expressed in the presence of the A6 PUFA elongase Substrates, 18:306 (Y-linolenic acid, GLA) and Des AS HDxxH QHxxxHH QxxHH 18:4c)3 (stearidonic acid, STA). GC analysis of the FAMEs of transformed yeast cells showed that PiELO1 elongated GLA (0179. Notably, cysteine (C) in the first his-box and the first and STA, converting them into dihomo-Y-linoleic acid histidine (H) in the third his-boxes, respectively, are con (DGLA, 20:3(O6) and eicosatetraenoic acid (20:4(O3), respec served only in A12 desaturases. The second residue in the first tively (FIG. 8). The yeast cells harboring the empty vector his-box of the all three types of desaturases is acidic; in A6 alone did not demonstrate any elongation activity on the and A5 desaturases it is mostly aspartic acid (D), and in A12 added substrates, confirming that the PiELO1 encoded desaturases mostly glutamic acid (E). This indicates the enzyme has a A6 PUFA elongase activity. Feeding the importance of an acidic residue at this position for desatura PiELO1 transformants with the Co6 fatty acids, LA and ARA, tion. Similarly to other A6 and A5 desaturases glutamine (Q) did not result in their elongation (not shown). is found in the third his-box of PiDesG and PiDes5 (FIG. 1; 0176 Real-time quantitative PCR was performed to quan Table 8) and in the second his-box of A5 desaturases. The titate the alterations in expression levels of the A6 PiELO1 in replacement of the H residue with Q in the third his-box of A6 P incisa cells under nitrogen starvation. The expression lev and A5 desaturases points to the role of Q in PUFA desatura els of the genes under nitrogen starvation were measured and tion. Indeed, replacing this Q with histidine or isoleucine normalized to the expression level of the endogenous control eliminated the enzyme activity of the recombinant A6 desatu gene 18S SSU rRNA. The fold change in the expression level rase in yeast cells. Glutamine was also found to be highly of the target genes in P incisa cells grown for 3, 7 and 14 don conserved in the third his-box of the A4 desaturases Pavlova N-free medium was calculated relative to the expression level lutheri (AY332747), Euglena gracilis (AY278558), and of the target genes in the log phase (time 0). The results Thraustochytrium sp. (AF489589). showed that during nitrogen starvation the mRNA of the 0180 Heterologous expression of PiDes6 and PiDes5 in PiELO1 gene was induced to its highest level at day 3 (14 fold yeast cells confirmed their A6 and A5 activity by conversion increase over time 0), decreasing thereafter to a level still of supplemented fatty acids to the corresponding desaturation higher than that of day 0 (FIG. 9). After 7 and 14 d, the products. PiDes 12 demonstrated very low desaturation activ expression level of the PiELO1 gene was still 7.5 and 4.3 fold ity, which could not be enhanced by the 5' modification of the higher, respectively. The level of expression of the PiELO1 inserted sequence. A similar low activity in yeast was also gene correlated with the increase in the share of ARA and the demonstrated in some cases, such as for A5 and A12 desatu C20/(C16+C18) elongation ratio (Table 7). The share of the rases of O. tauri and Chlorella vulgaris NJ-7, respectively. elongation product, DGLA, increased sharply at day 3 (50% 0181 PiDesG and PiDes5 desaturated both co3 and co6 increase over time 0) and decreased thereafter. fatty acids with similar efficiency (FIG. 3: Table 4). Various 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 18:3006 18:303 20:306 20:4006. 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)

0177. The capacity of P incisa to accumulate large quan results concerning (D3/cp6 Substrate preference were reported tities of ARA-rich TAG under nitrogen starvation, Suggested for functionally characterized A6 and A5 desaturases from that it would be of great interest to study its genes and different organisms that were expressed in yeast. A front-end enzymes involved in the accumulation of VLC-PUFA. In the PiDes5 desaturated its principal substrate 20:3()6 as well as present work, P incisa A12, A6, and A5 desaturases were 20:4c03; in addition, non-methylene interrupted fatty acids cloned and studied, which in conjunction with a recently were also produced as a result of its activity on 20:3 (03, and on cloned A6 specific PUFA elongase U. Iskandarov, I. Khozin both endogenous and exogenous 18:1, but with lower effi US 2013/0019341 A1 Jan. 17, 2013 24 ciency. PiDes5 produced 18:2^* from 18:1 but was more is a prerequisite for manipulating algal species to produce active when 18:1 was exogenously supplied. CrDES did Sustainable oils of pharmaceutical and neutraceutical values. insert A5 double bond on both 18:1 and 18:2 producing the 0185. A cDNA (PiELO1) of an elongase encoding for a non-methylene interrupted 18:2^' and 18:3^'', while deduced protein was isolated from P incisa, structurally simi adding a A7 double bond to 20:2006 and 20:30a). Apparently, lar to 46 PUFA elongase gene products from microalgae, in addition to the fatty acid chain length, the location and lower plants and fungi (FIG. 5). The deduced amino acid sequence of the PiELO1 ORF was about 50% identical to that number of double bond, and the form of the substrate (lipid of 46 elongases from the liverwort M. polymorpha or CoA bound) are also crucial for A5 desaturation. (AAT85662), the green marine microalga O. tauri PiDesG desaturated neither the yeast major monounsaturated (AAV67797) and the moss P. patens (AAL84174). In simi fatty acids nor the exogenously supplied 18:1. PiDesG did not larity to recently cloned PUFA elongases, the predicted pro act on 20:3c)3, indicating that it is specific for A9 C18 PUFA. tein is highly hydrophobic and has two strongly hydrophobic It appears that not only the organisms being transformed, but transmembrane regions, the first one located about 50 amino also the gene origin, determine the Substrate specificity of the acids downstream of the N-terminus and the secondone in the recombinant A6 and A5 desaturase. Functional characteriza vicinity of the C-terminus. The PiELO1 sequence was iden tion of PiDesG and PiDes5 confirmed the previously reported tified in a C-terminal lysine-rich motif, important for the Substrate specificities of these desaturases which were gen endoplannic reticulum targeting, as well as four conserved erally restricted to C18 and C20 substrates, respectively. motifs FYxSKXXEFxDT, QxxxLHVYHHXXI, NSXXH VXMYXYY and TxxQxxQF, including a highly conserved 0182. In Pincisa it was shown that PC and DGTS are used histidine box suggested to be functionally important for for lipid-linked C18 A6 desaturation whereas mostly PE is PUFA elongation (FIG. 5). These conserved motifs were not used for C20 A5 desaturation. PiDes5 featured higher sub found in other classes of plant microsomal elongases, B strate conversion rate in comparison to PiDesG. A relatively ketoacyl CoA synthases and fatty acid elongases (FAE) fast emergence of substantial percentage of ARA (10.6% involved in extraplastidial elongation of Saturated and conversion after 3 h of feeding) pursued us to study ARA monounsaturated fatty acids. A variant histidine box QAFHH distribution in individual lipid classes of the transformed with three replacements in C18-A9-PUFA elongase IgASE1 yeast (24 h of feeding). ARA was detected in all analyzed from I. galbana is thought to be important for enzymatic phospho- and neutral lipid classes of S. cerevisiae expressing activity rather then for substrate specificity. PiDes5 (Table 5). Similar conversion percentages were deter 0186 PiELO1 is another example of a single step A6 mined in all analyzed phospholipids, however, ARA distribu PUFA elongases cloned from an algal species. Similarly to tion showed preference for the major phospholipids, PC, GLELO of M. alpina, PiELO1 prefers the A6 C18 PUFA followed by PE. In P incisa, PE was found to be the main site substrates, GLA and STA. Only these A6 fatty acids were, for lipid-linked A5 desaturation C. Bigogno, I. Khozin when exogenously added, elongated to the respective prod Goldberg, D. Adlerstein, Z. Cohen, Biosynthesis of arachi ucts by S. cerevisiae cells transformed with PiELO1 (FIG. 8). donic acid in the oleaginous microalga Parietochloris incisa Transformation of a higher plant so as to render it to produce (Chlorophyceae): Radiolabeling studies, Lipids 37 (2002) A6 PUFA requires that the elongase used will have a high 209-216, while PC, a major A6 acyl lipid desaturation sub selectivity for A6 PUFA, thereby reducing the appearance of strate in this organism, was assumed to be utilized for A5 side products in the transformed plant. Bifunctional inverte desaturation, too. brate PUFA elongases with both A6 and A5 activities (Om ELO, XiELO, and CiELO) are less desirable in plant trans 0183. The quantitative RT-PCR results revealed that the formations. gene expression levels of PiDes 12, PiDesG, and PiDes5 fol 0187 Phylogenetic analysis showed (FIG. 6) that the lowed a similar pattern during the course of nitrogen starva PUFA elongases are not strictly divided according to their tion. The major transcriptional activation of the all three substrate specificity. The A6 elongases of algal (Ot...LO1, desaturases occurred on day 3 coinciding with a sharp rise in TpELO1, PiELO1) and moss (PpELO1) origin are function the percentage of ARA, which almost doubled (FIG.4, Table ally restricted to the elongation of A6-C18-PUFAs, however 6). The same expression pattern featured the P incisa A6 these elongases are placed in separate groups on the phylo PUFA elongase, however, at lower level. It was shown in genetic tree (FIG. 7). PiELO1 is closely related to OtELO1 radiolabeling and inhibitor studies, that ARA biosynthesis in isolated from a chloropyte and a lower plant rather than to P incisa follows the Co6 pathway. The concerted transcrip ELO1 genes isolated from a diatom, although both are spe tional activation of the PiDes12, PiDesG, PiDes5 and PiELO1 cific for the elongation of A6-C18-PUFAs (FIG. 7). PiELO1 genes was accompanied by an increase in the percent share of is highly similar to and is placed in the same group with both 18:1, a main chloroplast-derived fatty acid exported to ER A6 and A5 elongases of the liverwort M. polymorpha. and substrate of A12 desaturase. High expression of ARA Kajikawa et al. Suggested that MpELO2, a A5 elongase, is biosynthetic genes was accompanied by enhanced A5 and A6 likely to have originated through gene duplication of the desaturations (Table 6). MpELO1 gene. The algal A5 PUFA elongases, Ot...LO2, 0184. In conclusion, the A12, A6 and A5 fatty acid desatu TpELO2 and the Psalina ELO1 are more likely to share a rases involved in ARA biosynthesis in P incisa were identi common branch with the mammalian and animal A5 PUFA fied and functionally characterized. The corresponding ORFs elongases, OmELO and HsELO2, while they are also similar PiDes 12, PiDesG, and PiDes5, expressed in yeast confirmed to bifunctional PUFA elongases such as CiELO 1/2. their favorable enzymatic activity. Nitrogen starvation led to 0188 Quantitative real timd PCR studies revealed that the an increased transcription of the cloned genes reaching maxi expression level of the PiELO1 gene was up regulated during mum on day 3 and enhanced accumulation of ARA thereafter. the time course of N-starvation (FIG. 9). Nitrogen starvation Understanding the mechanisms underlying gene transcrip led to a continuous increase in the share of ARA and the tion regulation in metabolic pathways and characteristics of C20/(C16+C18) elongation ratio (Table 7). However, a major enzymes involved in ARA and lipid biosynthesis in P incisa transcriptional activation of PiELO1 which occurred on day 3 US 2013/0019341 A1 Jan. 17, 2013

(14-fold increase in transcript level) coincided with the steep 0189 The significance of the coordinated transcription rise in AA accumulation and elongation ratio (Table 7). The and action of desaturases and elongases in ARA biosynthesis increase in PiELO1 transcription level followed by enhanced in P incisa is yet to be elucidated. Possibly, the elongation of biosynthesis of ARA may be interpreted as an increase in GLA by PiELO1 could be rate-limiting in ARA biosynthesis PiELO1 enzyme level and/or enzymatic activity. The impor as it is in M. alpina. Abbadi et al. (2004) speculated that in tance of the transcriptional activation of PiELO1 is supported transgenic plants modified with VLC-PUFA biosynthesis by the fact that PUFA elongase was the only ARA biosynthe genes, Substrate availability rather than enzymatic activity is sis related gene that was obtained from the subtractive library. rate-limiting in the A6 elongation of PUFA.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 46

<21 Os SEQ ID NO 1 &211s LENGTH: 379 212s. TYPE: PRT <213> ORGANISM: P. 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 Lieu. Arg Lys Ala Ile Pro Wal His Cys Phe Glu Arg Ser Ile Pro Arg 35 4 O 45 Ser Phe Ala Tyr Lieu Ala Ala Asp Lieu Ala Ala Ile Ala Val Met Tyr SO 55 60

Tyr Lieu Ser Thr Phe Ile Asp His Pro Ala Val Pro Arg Wall Lieu Ala 65 70 7s

Trp Gly Lieu. Leu Trp Pro Ala Tyr Trp Tyr Phe Glin Gly Ala Wall Ala 85 90 95

Thr Gly Val Trp Val Ile Ala His Glu. Cys Gly His Glin Ala Phe Ser 1OO 105 110 Pro Tyr Glin Trp Lieu. Asn Asp Ala Val Gly Lieu Val Lieu. His Ser Cys 115 12O 125 Leu Lleu Val Pro Tyr Tyr Ser Trp Llys His Ser His Arg Arg His His 13 O 135 14 O Ser Asn Thr Gly Ser Thir Thr Lys Asp Glu Val Phe Val Pro Arg Glu 145 15 O 155 16 O

Ala Ala Met Val Glu Ser Asp Phe Ser Lieu Met Gln Thr Ala Pro Ala 1.65 17 O Arg Phe Leu Val Ile Phe Val Ser Lieu. Thir Ala Gly Trp Pro Ala Tyr 18O 185 190

Lieu. Phe Ala Asn Ala Ser Gly Arg Llys Tyr Gly Lys Trp Ala Asn His 195 2 OO 2O5

Phe Asp Pro Tyr Ser Pro Ile Phe Thr Lys Arg Glu Arg Ser Glu Ile 210 215 22 O

Val Val Ser Asp Val Ala Lieu. Thr Val Val Ile Ala Gly Lieu. Tyr Ser 225 23 O 235 24 O Lieu. Gly Lys Ala Phe Gly Trp Ala Trp Lieu Val Lys Glu Tyr Val Ile 245 25 O 255

Pro Tyr Lieu. Ile Val Asn Met Trp Lieu Val Met Ile Thr Lieu. Lieu. Glin 26 O 265 27 O His Thr His Pro Glu Lieu Pro His Tyr Ala Asp Lys Glu Trp Asp Trp 27s 28O 285 US 2013/0019341 A1 Jan. 17, 2013 26

- Continued Lieu. Arg Gly Ala Lieu Ala Thr Cys Asp Arg Ser Tyr Gly Gly Met Pro 29 O 295 3 OO Asp His Lieu. His His His Ile Ala Asp Thr His Val Ala His His Lieu 3. OS 310 315 32O Phe Ser Thr Met Pro His Tyr His Ala Glin Glu Ala Thr Glu Ala Ile 3.25 330 335 Llys Pro Ile Lieu. Gly Llys Tyr Tyr Lys Glin Asp Lys Arg Asn Val Trp 34 O 345 35. O Ala Ala Lieu. Trp Glu Asp Phe Ser Lieu. Cys Arg Tyr Val Ala Pro Asp 355 360 365 Thir Ala Gly Ser Gly Ile Lieu. Trp Phe Arg Ala 37 O 375

<210s, SEQ ID NO 2 &211s LENGTH: 48O 212. TYPE: PRT <213> ORGANISM: P. incisa

<4 OOs, SEQUENCE: 2 Met Cys Glin Gly Glin Ala Val Glin Gly Lieu. Arg Arg Arg Ser Ser Phe 1. 5 1O 15 Lieu Lys Lieu. Thr Gly Asp Ala Ile Lys Gly Ala Val Ala Ala Ile Ser 2O 25 3O Asp Phe Asn Llys Lieu Pro Ala Ala Thr Pro Val Phe Ala Arg Arg Ser 35 4 O 45 Lieu. Ser Asp Ser Ala Lieu. Glin Glin Arg Asp Gly Pro Arg Ser Lys Glin SO 55 6 O Glin Val Thir Lieu. Glu Glu Lieu Ala Gln His Asn Thr Pro Glu Asp Cys 65 70 7s 8O Trp Lieu Val Ile Lys Asn Llys Val Tyr Asp Val Ser Gly Trp Gly Pro 85 90 95 Gln His Pro Gly Gly His Val Ile Tyr Thr Tyr Ala Gly Lys Asp Ala 1OO 105 11 O Thr Asp Val Phe Ala Cys Phe His Ala Glin Thr Thr Trp Ser Gln Leu 115 12 O 125 Arg Pro Phe Cys Ile Gly Asp Ile Val Glu Glu Glu Pro Met Pro Ala 13 O 135 14 O Lieu. Lieu Lys Asp Phe Arg Glu Lieu. Arg Thr Arg Lieu. Glin Glin Glin Gly 145 150 155 160 Lieu. Phe Arg Ser Asn Llys Lieu. Tyr Tyr Lieu. Tyr Llys Val Ala Ser Thr 1.65 17O 17s Lieu. Ser Lieu. Lieu Ala Ala Ala Lieu Ala Val Lieu. Ile Thr Glin Arg Asp 18O 185 19 O Ser Trp Lieu. Gly Lieu Val Gly Gly Ala Phe Lieu. Lieu. Gly Lieu. Phe Trp 195 2OO 2O5 Gln Glin Ser Gly Trp Leu Ala His Asp Phe Lieu. His His Glin Val Phe 21 O 215 22O Thir Asp Arg Glin Trp Asn. Asn. Wal Met Gly Tyr Phe Lieu. Gly Asn. Wall 225 23 O 235 24 O Cys Glin Gly Phe Ser Thr Asp Trp Trp Llys Ser Lys His Asn Val His 245 250 255 His Ala Val Pro Asn. Glu Lieu. Asp Ser Asp Ser Lys Ala Ala Arg Asp 26 O 265 27 O US 2013/0019341 A1 Jan. 17, 2013 27

- Continued Pro Asp Ile Asp Thir Lieu Pro Lieu. Lieu Ala Trp Ser Ser Glu Met Lieu. 27s 28O 285 Asp Ser Met Ser Asn. Ser Gly Ala Arg Lieu. Phe Val Arg Met Gln His 29 O 295 3 OO Tyr Phe Phe Phe Pro Ile Lieu. Leu Phe Ala Arg Met Ser Trp Cys Glin 3. OS 310 315 32O Glin Ser Val Ala His Ala Ser Asp Lieu. Ser Arg Thr Ser Lys Ala Gly 3.25 330 335 Val Tyr Glu Lieu Ala Tyr Lieu Ala Lieu. His Tyr Ala Trp Phe Lieu. Gly 34 O 345 35. O Ala Ala Phe Ser Val Lieu Pro Pro Lieu Lys Ala Val Val Phe Ala Lieu 355 360 365 Lieu. Ser Gln Met Phe Ser Gly Phe Lieu. Leu Ser Ile Val Phe Val Glin 37 O 375 38O Ser His Asn Gly Met Glu Val Tyr Ser Asp Thr Lys Asp Phe Val Thr 385 390 395 4 OO Ala Glin Ile Val Ser Thr Arg Asp Ile Lieu. Ser Asn Val Trp Asn Asp 4 OS 41O 415 Trp Phe Thr Gly Gly Lieu. Asn Tyr Glin Ile Glu. His His Leu Phe Pro 42O 425 43 O Thir Lieu Pro Arg His Asn Lieu. Gly Llys Val Glin Llys Ser Ile Met Glu 435 44 O 445 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: P. 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 12 O 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 US 2013/0019341 A1 Jan. 17, 2013 28

- Continued 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 Glu Phe Val Thr Gly Trp Met Leu Ala Phe Met Phe Glin Val Ala His 355 360 365 Val Thir Ser Asp Val Ser Tyr Lieu. Glu Ala Asp Llys Thr Gly Llys Val 37 O 375 38O Pro Arg Gly Trp Ala Ala Ala Glin Ala Ala Thir Thr Ala Asp Phe Ala 385 390 395 4 OO His Gly Ser Trp Phe Trp Thr Glin Ile Ser Gly Gly Lieu. Asn Tyr Glin 4 OS 41O 415 Val Val His His Leu Phe Pro Gly Ile Cys His Lieu. His Tyr Pro Ala 42O 425 43 O Ile Ala Pro Ile Val Lieu. Asp Thr Cys Lys Glu Phe Asn Val Pro Tyr 435 44 O 445 His Val Tyr Pro Thr Phe Val Arg Ala Leu Ala Ala His Phe Llys His 450 45.5 460 Lieu Lys Asp Met Gly Ala Pro Thr Ala Ile Pro Ser Lieu Ala Thr Val 465 470 47s 48O Gly

<210s, SEQ ID NO 4 &211s LENGTH: 114 O &212s. TYPE: DNA <213> ORGANISM: P. incisa

<4 OOs, SEQUENCE: 4 atggggaaag gaggctgtta C caggc.cggg cct cotagog caaagaaatg ggagagtagg 6 O gtgcc.cactg ccaaac ccga gttcacgatc ggaac cct co goaaagctat accogg to cac 12 O tgct tcgaac ggtc.catc.cc ticggit cottt gcc taccttg C9gcagacct ggcggctatt 18O gcggtcatgt act acctgag cactitt catc gat catc.ccg ccgtgcc.gcg ggtc.ctggCC 24 O

US 2013/0019341 A1 Jan. 17, 2013 31

- Continued

Met Ala Lieu. Thir Ala Ala Trp His Llys Tyr Asp Ala Ile Val Ser Arg

Phe Val Phe Asp Gly Lieu. Arg Arg Val Gly Lieu. Glin Glu Ile Glin Gly 2O 25 3O

His Pro Ser Wall Ile Thir Ala His Lieu. Pro Phe Ile Ala Ser Pro Thr 35 4 O 45 Pro Glin Val Thr Phe Val Lieu Ala Tyr Lieu. Lieu. Ile Val Val Cys Gly SO 55 6 O Val Ala Ala Lieu. Arg Thr Arg Llys Ser Ser Ala Pro Arg Glu Asp Pro 65 70 7s 8O Ala Trp Lieu. Arg Lieu. Lieu Val Glin Ala His Asn Lieu Val Lieu. Ile Ser 85 90 95 Lieu. Ser Ala Tyr Met Ser Ser Ala Ala Cys Tyr Tyr Ala Trp Llys Tyr 1OO 105 11 O Gly Tyr Arg Phe Trp Gly Thr Asn Tyr Ser Pro Lys Glu Arg Asp Met 115 12 O 125 Gly Gly Lieu. Ile Tyr Thr Phe Tyr Val Ser Lys Lieu. Tyr Glu Phe Val 13 O 135 14 O Asp Thir Lieu. Ile Met Lieu Lleu Lys Gly Llys Val Glu Glin Val Ser Phe 145 150 55 160 Lieu. His Val Tyr His His Ala Ser Ile Ser Thr Ile Trp Trp Ala Ile 1.65 17O 17s Ala Tyr Val Ala Pro Gly Gly Asp Ala Trp Tyr Cys Cys Phe Lieu. Asn 18O 185 19 O Ser Leu Val His Val Lieu Met Tyr Thr Tyr Tyr Lieu. Leu Ala Thr Lieu. 195 2OO 2O5 Lieu. Gly Lys Asp Ala Lys Ala Arg Arg Llys Tyr Lieu. Trp Trp Gly Arg 21 O 215 22O Tyr Lieu. Thr Glin Phe Gln Met Phe Glin Phe Val Thr Met Met Leu Glu 225 23 O 235 24 O Ala Ala Tyr Thr Trp Ala Tyr Ser Pro Tyr Pro Llys Phe Leu Ser Lys 245 250 255 Lieu. Leu Phe Phe Tyr Met Ile Thr Lieu. Leu Ala Leu Phe Ala Asn Phe 26 O 265 27 O Tyr Ala Glin Llys His Gly Ser Ser Arg Ala Ala Lys Glin Llys Lieu. Glin 27s 28O 285

<210s, SEQ ID NO 8 &211s LENGTH: 867 &212s. TYPE: DNA <213> ORGANISM: P. incisa

<4 OOs, SEQUENCE: 8 atggcattga C9gcggcctg gcacalagtac gacgctatcg ttagt cqctt tdtttitcgat 6 O ggcttgcgca gggttggcct gcaa.gagatt Caaggcc acc cct cqgtgat Caccgcc cac 12 O citt.ccct tca tagcct cocc aacgc.cacaa gtgacgttcg togctggcct a tictogctgatt 18O gttgtctg.cg gggttgcc.gc. tctg.cgitacg agaaagt cqt ccgcaccitcg cgaggat.ccg 24 O gcgtggctgc gactgcttgt gcaa.gc.gcac aacttggtgc taatcagcct tagcgc.ctac 3OO atgtcCtctg. cc.gc.ctgcta ctatgcttgg aaatacggct at aggttittggggcacaaac 360 tatagc.ccca aggagcggga catgggaggg ct catctata cct tttacgt gtcCaagctg 42O US 2013/0019341 A1 Jan. 17, 2013 32

- Continued tacgagtttgttggatacgct gat catgctg Ctcaagggca aggtggagca ggtttcttitt 48O ttgcacgt.ct accaccacgc t to catat co acgatctggit gggcaatcgc atacgt.cgca 54 O Cctggtggtg acgc.ctggta ctgctgctitc ctgaact cqc tggtccacgt. act catgitac acatact acc togcttgcgac gctgctggga aaggacgc.ca agg.cgcggcg caagtatttg 660 tggtggggac gctacct cac t cagttccag atgttccagt ttgttgacgat gatgctic gag 72 O gcagcgtaca Cttgggccta ctic toccitac cc caagttitt tat caaagct gctgttctitt tacatgat ca citctgttggc cctgtttgca aacttictato Cacagaa.gca tggcagcagc 84 O cgggcagc.ca agcaaaagct gcagtaa 867

SEO ID NO 9 LENGTH: 30 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: forward primer used for partial sequence of delta 12 desatuarase

SEQUENCE: 9 Cys Ala Cys Met Tyr Cys Val Thr Ser Thr His Cys Val Trp Gly Cys 1. 5 15 Thr Gly Cys Thr Gly Val Trp Asix Cys Cys Cys Cys Ala Cys 25

SEQ ID NO 10 LENGTH: 27 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Reverse primer for partial sequence of delta 12 desaturase

SEQUENCE: 1.O

Ctgc.ccgaag ttgaccgcgg C9tgctg 27

SEQ ID NO 11 LENGTH: 21 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Forward primer for partial sequence of delta 6 desaturase

SEQUENCE: 11 Thr Gly Gly. Thr Gly Gly Ala Ala Arg Cys Ala Tyr Ala Ala Arg Cys 1. 5 15 Ala Tyr Ala Ala Tyr

SEQ ID NO 12 LENGTH: 30 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Reverse primer for partial sequence of delta 6 desaturase

SEQUENCE: 12 Gly Cys Gly Ala Gly Gly Gly Ala Thr Cys Cys Ala Ala Gly Gly Arg 1. 5 15 US 2013/0019341 A1 Jan. 17, 2013 33

- Continued Ala Ala Asn Ala Arg Arg Thr Gly Arg Thr Gly Tyr Thr Cys 2O 25 3O

<210s, SEQ ID NO 13 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for partial sequence of delta 5 desaturase

<4 OOs, SEQUENCE: 13 Ala Thr His Arg Ala Ile Gly Arg Ile Ala Ala Arg Gly. Thir Ile Thr 1. 5 1O 15 Ala Tyr Gly Ala Tyr Gly Thr 2O

<210s, SEQ ID NO 14 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for partial sequence of delta 5 desaturase

<4 OOs, SEQUENCE: 14 Gly Gly Ile Ala Tyr Ile Llys Trp Ile Thr Ser Asp Ala Thr Arg Thr 1. 5 1O 15 Cys Ile Gly Gly Arg Thr Cys 2O

<210s, SEQ ID NO 15 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for partial sequence of Actin

<4 OOs, SEQUENCE: 15 agatctggca ccacacct to ttca 24

<210s, SEQ ID NO 16 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for partial sequence of Actin <4 OOs, SEQUENCE: 16 tgttgttgta gaggtoctitg cgga 24

<210s, SEQ ID NO 17 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for RACE amplification of delta 12 desaturase

<4 OOs, SEQUENCE: 17 C Cacat agcg gcacaggctgaaatc 25

<210s, SEQ ID NO 18 &211s LENGTH: 25 US 2013/0019341 A1 Jan. 17, 2013 34

- Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for RACE amplification of delta 12 desaturase

<4 OOs, SEQUENCE: 18 gctctgggag gattt Cagcc ttgc 25

<210s, SEQ ID NO 19 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for RACE amplification of delta 6 desaturase

<4 OOs, SEQUENCE: 19 gacacaatct gggc.cgtcac aaagtic 26

<210s, SEQ ID NO 2 O &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for RACE amplification of delta 6 desaturase

<4 OOs, SEQUENCE: 2O ggactttgttg acggcc.ca.ga ttgttgtc. 27

<210s, SEQ ID NO 21 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for RACE amplification of delta 5 desaturase

<4 OOs, SEQUENCE: 21 actgaccctic citctgtgtcc tictitcg 26

<210s, SEQ ID NO 22 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for RACE amplification of delta 5 desaturase

<4 OOs, SEQUENCE: 22 tgitacgc.caa gtc.gct gacc atcc 24

<210s, SEQ ID NO 23 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for full length cloning of delta 12 desaturase

<4 OOs, SEQUENCE: 23 tgga attcaa aatggggaaa ggaggctg 28

<210s, SEQ ID NO 24 US 2013/0019341 A1 Jan. 17, 2013 35

- Continued

&211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for full-length cloning of delta 12 desaturase

<4 OOs, SEQUENCE: 24 Ctgtctagat Caagcgcgga accacagg 28

<210s, SEQ ID NO 25 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for full-length cloning of delta 6 desaturase

<4 OOs, SEQUENCE: 25 tcga attcaa aatgtgc.cag gga cagg 27

<210s, SEQ ID NO 26 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for full-length cloning of delta 6 desaturase

<4 OOs, SEQUENCE: 26 ggct ct agac taggccticag Ctgcc acg 28

<210s, SEQ ID NO 27 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for full-length cloning of delta 5 desaturase

<4 OOs, SEQUENCE: 27 cCaaagctta aaatgatggc tigtaiacaga 29

<210s, SEQ ID NO 28 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for full-length cloning of delta 5 desaturase

<4 OOs, SEQUENCE: 28 gctictagact atc.ccacggit ggc.ca 25

<210s, SEQ ID NO 29 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse gene specific primer for putative PUFA elongase

<4 OOs, SEQUENCE: 29 cc.cggctgct gcc atgct tc ttg 24 US 2013/0019341 A1 Jan. 17, 2013 36

- Continued <210s, SEQ ID NO 3 O &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse gene specific primer for putative PUFA elongase

<4 OOs, SEQUENCE: 30 tgggg taggg agagtaggcc Caagt 25

<210s, SEQ ID NO 31 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward gene specific primer for putative PUFA elongase

<4 OOs, SEQUENCE: 31 gcct acatgt cct ctd.ccgc ctdcta 26

<210s, SEQ ID NO 32 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward gene specific primer for putative PUFA elongase

<4 OOs, SEQUENCE: 32 gcggga catg ggagggct catct at acc 28

<210s, SEQ ID NO 33 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for P. incisa ELO1 ORF <4 OOs, SEQUENCE: 33 agga attcaa aatggcattg acggcggc ct 3 O

<210s, SEQ ID NO 34 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for P. incisa ELO1 ORF <4 OOs, SEQUENCE: 34 cattctagat tactgcagct tttgcttggc tigc 33

<210s, SEQ ID NO 35 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for real-time quantitative PCR of P. incisa ELO1

<4 OOs, SEQUENCE: 35 aagctgtacg agtttgttgga tacgct 26

<210s, SEQ ID NO 36 US 2013/0019341 A1 Jan. 17, 2013 37

- Continued

&211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for Real-time quantitative PCR for P. incisa ELO1

<4 OOs, SEQUENCE: 36 ggatatggala gC9tggtggit aga 23

<210s, SEQ ID NO 37 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for real-time quantitative PCR of 18S SSU rRNA

<4 OO > SEQUENCE: 37 tgaaagacga acttctgcga aagca 25

<210s, SEQ ID NO 38 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for real-time quantitative PCR of 18S SSU rRNA

<4 OOs, SEQUENCE: 38 agtcggcatc gtt tatggitt gaga 24

<210s, SEQ ID NO 39 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for real-time quantitative PCR of P. incisa delta 12 desaturase

<4 OOs, SEQUENCE: 39 gaag caccac Caaggatgag git 22

<210s, SEQ ID NO 4 O &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for real-time quantitative PCR of P. incisa delta 12 desaturase

<4 OOs, SEQUENCE: 4 O agcgagacga agatgaccag gaa 23

<210s, SEQ ID NO 41 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for real-time quantitative PCR of P. incisa delta 6 desaturase

<4 OOs, SEQUENCE: 41 US 2013/0019341 A1 Jan. 17, 2013 38

- Continued actitcc tigca ccaccagg to titc 23

<210s, SEQ ID NO 42 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for real-time quantitative PCR of P. incisa delta 6 desaturase

<4 OOs, SEQUENCE: 42 tcqtgcttgc tict tccacca git 22

<210s, SEQ ID NO 43 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for real-time quantitative PCR of P. incisa delta 5 desaturase

<4 OOs, SEQUENCE: 43 taagtgc.cag ggctgtgcta ga 22

<210s, SEQ ID NO 44 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for real-time quantitative PCR of P. incisa delta 5 desaturase

<4 OOs, SEQUENCE: 44 gaactgaccc ticcitctgttgt cct 23

<210s, SEQ ID NO 45 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward primer for real-time quantitative PCR of P. incisa Actin

<4 OOs, SEQUENCE: 45 cgtc.ca.gctic cacgattgag aaga 24

<210s, SEQ ID NO 46 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse primer for real-time quantitative PCR of P. incisa Actin

<4 OOs, SEQUENCE: 46 atggagttga aggcggtctic gt 22

1. An isolated protein comprising, an amino acid sequence 4. A composition comprising the protein of claim 3. set forth in SEQED NO: 1. 2. A composition comprising the protein of claim 1. 5. An isolated protein comprising, an amino acid sequence 3. An isolated protein comprising, an amino acid sequence set forth in SEQID NO:3. set forth in SEQID NO: 2. 6. A composition comprising the protein of claim 5. US 2013/0019341 A1 Jan. 17, 2013 39

7. An isolated polynucleotide comprising a coding portion 30. The method of claim 25, wherein said plant, said plant encoding the protein of claim 1. cell, or said alga is grown under oleogenic conditions, under 8. The isolated polynucleotide of claim 7, wherein said nitrogen starvation conditions, or a combination thereof. coding portion comprises a nucleic acid sequence set forth in 31. The method of claim 25, wherein said producing very SEQID NO: 4. long-chain polyunsaturated fatty acid (VLC-PUFA) is 9. An isolated polynucleotide comprising a coding portion enhancing oil storage, arachidonic acid accumulation, eicosa pentaenoic acid accumulation, docosahexaenoic acid accu encoding the protein of claim 3. mulation, dihomo-gamma-linolenic acid accumulation, or a 10. The isolated polynucleotide of claim 9, wherein said combination thereof. coding portion comprises a nucleic acid sequence set forth in 32. The method of claim 25, further comprising the a step SEQID NO: 5. of transforming said plant, said plant cell, or said alga with a 11. An isolated polynucleotide comprising a coding por polynucleotide of claim 9, a polynucleotide of claim 11, a tion encoding the protein of claim 5. polynucleotide encoding a PUFA-specific elongase, or any 12. The isolated polynucleotide of claim 11, wherein said combination thereof. coding portion comprises a nucleic acid sequence set forth in 33. The method of claim 26, further comprising the a step SEQID NO: 6. of transforming said plant, said plant cell, or said alga with a 13. An expression vector comprising the polynucleotide of polynucleotide of claim 7, a polynucleotide of claim 11, a claim 7. polynucleotide encoding a PUFA-specific elongase, or any 14. An expression vector comprising the polynucleotide of combination thereof. claim 9. 34. The method of claim 27, further comprising the a step 15. An expression vector comprising the polynucleotide of of transforming said plant, said plant cell, or said alga with a polynucleotide of claim 7, a polynucleotide of claim 9, a claim 11. polynucleotide encoding a PUFA-specific elongase, or any 16. A cell comprising the expression vector of claim 13. combination thereof. 17. A cell comprising the expression vector of claim 14. 35. The method of claim 26, wherein said plant, said plant 18. A cell comprising the expression vector of claim 15. cell, or said alga comprises linoleic acid (LA; 18:2006), C-li 19. A transgenic plant, a transgenic seed, or a transgenic nolenic acid (ALA; 18:3c)3), oleic acid (18:1), dihomo alga transformed by a polynucleotide of claim 7. gamma-linolenic acid (20:3c)6), phosphatidylcholine (PC), 20. A transgenic plant, a transgenic seed, or a transgenic diacylglyceroltrimethylhomoserine (DGTS), phosphatidyle alga transformed by a polynucleotide of claim 9. thanolamine (PE), or any combination thereof. 21. A transgenic plant, a transgenic seed, or a transgenic 36. The method of claim 26, wherein said VLC-PUFA is alga transformed by a polynucleotide of claim 11. eicosapentaenoic acid (EPA, 20:5c)3), docosahexaenoic acid 22. A transgenic seed, produced by a transgenic plant trans (DHA, 22:603), dihomo-gamma-linolenic acid (DGLA), or formed by a polynucleotide of claim 7. arachidonic acid (ARA, 20:4(O6) 23. A transgenic seed, produced by a transgenic plant trans 37. The method of claim 26, wherein said plant, said plant formed by a polynucleotide of claim 9. cell, or said alga is grown under oleogenic conditions, under 24. A transgenic seed, produced by a transgenic plant trans nitrogen starvation conditions, or a combination thereof. formed by a polynucleotide of claim 11. 38. The method of claim 26, wherein said producing very 25. A method of producing very long-chain polyunsatu long-chain polyunsaturated fatty acid (VLC-PUFA) is rated fatty acid (VLC-PUFA) in a plant, a plant cell, or an alga enhancing oil storage, arachidonic acid accumulation, eicosa comprising the step of transforming a plant, a plant cell, oran pentaenoic acid accumulation, docosahexaenoic acid accu alga with a polynucleotide of claim 7, thereby producing a mulation, dihomo-gamma-linolenic acid accumulation, or a VLC-PUFA in a plant, a plant cell, or an alga. combination thereof. 26. A method of producing very long-chain polyunsatu 39. The method of claim 27, wherein said plant, said plant rated fatty acid (VLC-PUFA) in a plant, a plant cell, or an cell, or said alga comprises linoleic acid (LA; 18:2006), a-li alga, comprising the step of transforming a plant, a plant cell, nolenic acid (ALA; 18:3c)3), oleic acid (18:1), dihomo oran alga with a polynucleotide of claim 9, thereby producing gamma-linolenic acid (20:3c)6), phosphatidylcholine (PC), a VLC-PUFA in a plant, a plant cell, or an alga. diacylglyceroltrimethylhomoserine (DGTS), phosphatidyle 27. A method of producing very long-chain polyunsatu thanolamine (PE), or any combination thereof. rated fatty acid (VLC-PUFA) in a plant, a plant cell, or an 40. The method of claim 27, wherein said VLC-PUFA is alga, comprising the step of transforming a plant, a plant cell, eicosapentaenoic acid (EPA, 20:5c)3), docosahexaenoic acid or an alga with a polynucleotide of claim 11, thereby produc (DHA, 22:603), dihomo-gamma-linolenic acid (DGLA), or ing a VLC-PUFA in a plant, a plant cell, or an alga. arachidonic acid (ARA, 20:4(O6) 28. The method of claim 25, wherein said plant, said plant 41. The method of claim 27, wherein said plant, said plant cell, or said alga comprises linoleic acid (LA; 18:2006), C-li cell, or said alga is grown under oleogenic conditions, under nolenic acid (ALA; 18:3c)3), oleic acid (18:1), dihomo nitrogen starvation conditions, or a combination thereof. gamma-linolenic acid (20:3c)6), phosphatidylcholine (PC), 42. The method of claim 27, wherein said producing very diacylglyceroltrimethylhomoserine (DGTS), phosphatidyle long-chain polyunsaturated fatty acid (VLC-PUFA) is thanolamine (PE), or any combination thereof. enhancing oil storage, arachidonic acid accumulation, eicosa 29. The method of claim 25, wherein said VLC-PUFA is pentaenoic acid accumulation, docosahexaenoic acid accu eicosapentaenoic acid (EPA, 20:5c)3), docosahexaenoic acid mulation, dihomo-gamma-linolenic acid accumulation, or a (DHA, 22:603), dihomo-gamma-linolenic acid (DGLA), or combination thereof. arachidonic acid (ARA, 20:4(O6)