Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis
2004; Elsevier BV; Volume: 45; Issue: 10 Linguagem: Inglês
10.1194/jlr.m400181-jlr200
ISSN1539-7262
AutoresAstrid Meyer, Helene Kirsch, Frédéric Domergue, Amine Abbadi, Petra Sperling, Jörg Bauer, Petra Cirpus, T. Zank, Hervé Moreau, Thomas Roscoe, Ulrich Zähringer, Ernst Heinz,
Tópico(s)Fatty Acid Research and Health
ResumoIn algae, the biosynthesis of docosahexaenoic acid (22:6ω3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5ω3; EPA) to 22:5ω3, which is required as a substrate for the final Δ4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Δ6-)C18-PUFAs and one specific for (Δ5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Δ6- and Δ5-elongases from T. pseudonana and O. tauri, respectively, with the Δ5- and Δ4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast.This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops. In algae, the biosynthesis of docosahexaenoic acid (22:6ω3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5ω3; EPA) to 22:5ω3, which is required as a substrate for the final Δ4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of (Δ6-)C18-PUFAs and one specific for (Δ5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the Δ6- and Δ5-elongases from T. pseudonana and O. tauri, respectively, with the Δ5- and Δ4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops. Docosahexaenoic acid (DHA) is a fatty acid with 22 carbon atoms and 6 methylene-interrupted Z-double bonds (22:6Δ4,7,10,13,16,19). Some human tissues such as brain, testis, and retina are characterized by membrane lipids carrying high proportions of this long-chain polyunsaturated fatty acid (LCPUFA), and many clinical studies have pointed out the crucial role of DHA in the development and functions of these tissues (1Muskiet F.A. Fokkema M.R. Schaafsma A. Boersma E.R. Crawford M.A. Is docosahexaenoic acid (DHA) essential? Lessons from DHA status regulation, our ancient diet, epidemiology and randomized controlled trials.J. Nutr. 2004; 134: 183-186Crossref PubMed Scopus (102) Google Scholar). In addition, DHA as well as the other LCPUFAs arachidonic acid (ARA; 20:4Δ5,8,11,14) and eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) are precursors of different classes of eicosanoid effectors involved in the regulation of many important functions in mammals. In view of the major roles attributed to LCPUFAs in human physiology, the reactions contributing to their biosynthesis have recently attracted growing interest (2Abbadi A. Domergue F. Meyer A. Riedel K. Sperling P. Zank T.K. Heinz E. Transgenic oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty acids?.Eur. J. Lipid Sci. Technol. 2001; 103: 106-113Crossref Scopus (29) Google Scholar). Because of its relevance for human nutrition, the biosynthetic sequence known in most detail is that realized in the mammalian liver and known as the Sprecher pathway (3Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. Acta. 2000; 1486: 219-231Crossref PubMed Scopus (656) Google Scholar). Moreover, LCPUFA biosynthesis was also studied in various organisms from phylogenetically divergent groups, such as algae, fungi, and lower plants. DHA is synthesized de novo in several microalgae as well as in some fungi, whereas in mammals its synthesis starts from the essential fatty acid α-linolenic acid (ALA; 18:3Δ9,12,15). Another route for DHA biosynthesis that will not be further described here is relying on polyketide synthase systems. It is found in some marine bacteria and primitive eukaryotes like the thraustochytrid protist Schizochytrium (4Metz J.G. Roessler P. Facciotti D. Levering C. Dittrich F. Lassner M. Valentine R. Lardizabal K. Domergue F. Yamada A. Yazawa K. Knauf V. Browse J. Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes.Science. 2001; 293: 290-293Crossref PubMed Scopus (553) Google Scholar). The currently best known routes for DHA biosynthesis are depicted in simplified form in Fig. 1. In the liver of mammals, a Δ6-desaturase, a Δ6-elongase, and a Δ5-desaturase successively convert ALA via stearidonic acid (STA; 18:4Δ6,9,12,15) and 20:4Δ8,11,14,17 to EPA in the so-called ω3 pathway. In parallel, the same set of enzymes accepts the other essential fatty acid linoleic acid (LA; 18:2Δ9,12) to form ARA in the ω6 pathway. It is important to note that because of the absence of an ω3-desaturase in mammals, the intermediates of the ω6 and ω3 pathways are not interconvertible. In mammalian liver, EPA is then elongated twice without an intervening desaturation. Recent data suggest that the same elongase (ELOVL2) accepts EPA as well as Δ7-22:5 (i.e., the reaction product of its own first elongation cycle), leading to Δ9-C24:5 (5de Antueno R.J. Knickle L.C. Smith H. Elliot M.L. Allen S.J. Nwaka S. Winther M.D. Activity of human Δ5 and Δ6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.FEBS Lett. 2001; 509: 77-80Crossref PubMed Scopus (100) Google Scholar). At this point, the Δ6-desaturase that is responsible for the formation of Δ6-C18-PUFAs gets involved a second time and inserts a Δ6 double bond in Δ9-C24:5, leading to the synthesis of Δ6-C24:6 (5de Antueno R.J. Knickle L.C. Smith H. Elliot M.L. Allen S.J. Nwaka S. Winther M.D. Activity of human Δ5 and Δ6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.FEBS Lett. 2001; 509: 77-80Crossref PubMed Scopus (100) Google Scholar, 6D'Andrea S. Guillou H. Jan S. Catheline D. Thibault J-N. Bouriel M. Rioux V. Legrand P. The same rat Δ6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis.Biochem. J. 2002; 364: 49-55Crossref PubMed Scopus (97) Google Scholar). Finally, Δ6-C24:6 is transferred from the endoplasmic reticulum membranes to the peroxisomes for one round of β-oxidative chain shortening and release of DHA. These final steps, from EPA to DHA, are attributable to the absence of a Δ4-desaturase activity in mammals and are characteristic for the Sprecher pathway. Labeling studies (7Buzzi M. Henderson R.J. Sargent J.R. Biosynthesis of docosahexaenoic acid in trout hepatocytes proceeds via 24-carbon intermediates.Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1997; 116: 263-267Crossref PubMed Scopus (95) Google Scholar) have shown that this sequence is also present in fish, suggesting that this route for DHA biosynthesis is not restricted to mammals. Up to ARA and EPA, the biosynthetic pathways found in mosses, fungi, and algae are basically identical to the mammalian sequence. Also in these organisms, two front-end desaturases (Δ6 and Δ5) and one Δ6-elongase convert either LA or ALA into ARA or EPA, respectively, in the ω6 and ω3 pathways (Fig. 1A). In addition, some algae can use an alternative pathway, in which LA and ALA are elongated to the corresponding Δ11-C20-PUFA (Fig. 1B), necessitating a subsequent Δ8-desaturation (8Qi B. Beaudoin F. Fraser T. Stobart A.K. Napier J.A. Lazarus C.M. Identification of a cDNA encoding a novel C18-Δ9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana.FEBS Lett. 2002; 510: 159-165Crossref PubMed Scopus (115) Google Scholar). Because many of these organisms possess an ω3-desaturase, they do not show the strict separation of ω6 and ω3 pathways typical for mammals. In addition, these organisms may use acyl chains of phospholipids as substrates for the desaturation reactions, whereas acyl-CoA thioesters are required for elongation reactions (9Domergue F. Abbadi A. Ott C. Zank T.K. Zähringer U. Heinz E. Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast.J. Biol. Chem. 2003; 278: 35115-35126Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In contrast, in mammals all of the intermediates are kept in the form of acyl-CoA thioesters. Most importantly for this study, the primary de novo producers of DHA appear to follow a simplified route for DHA synthesis in which a Δ5-elongase and a Δ4-desaturase are responsible for the conversion of EPA to DHA (10Qiu X. Hong H. MacKenzie S.L. Identification of a Δ4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea.J. Biol. Chem. 2001; 276: 31561-31566Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 11Meyer A. Cirpus P. Ott C. Schlecker R. Zähringer U. Heinz E. Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Δ4-fatty acyl group desaturase.Biochemistry. 2003; 42: 9779-9788Crossref PubMed Scopus (74) Google Scholar, 12Tonon T. Harvey D. Larson T.R. Graham I.A. Identification of a very long chain polyunsaturated fatty acid Δ4-desaturase from the microalga Pavlova lutheri.FEBS Lett. 2003; 553: 440-444Crossref PubMed Scopus (76) Google Scholar). Elongase complexes comprise four activities: the β-ketoacyl-CoA synthase, the ketoacyl-CoA reductase, the hydroxyacyl-CoA dehydratase, and the enoyl-CoA reductase. From these, the first enzyme is considered to be rate limiting and specificity controlling with regard to chain length and pattern of double bonds. ELO-type sequences involved in LCPUFA biosynthesis were cloned from the moss Physcomitrella patens (13Zank T.K. Zähringer U. Beckmann C. Pohnert G. Boland W. Holtorf H. Reski R. Lerchl J. Heinz E. Cloning and functional characterisation of an enzyme involved in the elongation of Δ6-polyunsaturated fatty acids from the moss Physcomitrella patens.Plant J. 2002; 31: 255-268Crossref PubMed Scopus (92) Google Scholar), the fungus Mortierella alpina (14Parker-Barnes J.M. Das T. Bobik E. Leonard A.E. Thurmond J.M. Chaung L-T. Huang Y-S. Mukerji P. Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids.Proc. Natl. Acad. Sci. USA. 2000; 97: 8284-8289Crossref PubMed Scopus (137) Google Scholar), the nematode Caenorhabditis elegans (15Beaudoin F. Michaelson L.V. Hey S.J. Lewis M.J. Shewry P.R. Sayanova O. Napier J.A. Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.Proc. Natl. Acad. Sci. USA. 2000; 97: 6421-6426Crossref PubMed Scopus (133) Google Scholar), the alga Isochrysis alpina (8Qi B. Beaudoin F. Fraser T. Stobart A.K. Napier J.A. Lazarus C.M. Identification of a cDNA encoding a novel C18-Δ9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana.FEBS Lett. 2002; 510: 159-165Crossref PubMed Scopus (115) Google Scholar), and different mammals [for a recent review, see ref. (16Leonard A.E. Pereira S.L. Sprecher H. Huang Y-S. Elongation of long-chain fatty acids.Prog. Lipid Res. 2004; 43: 36-54Crossref PubMed Scopus (431) Google Scholar)]. Because of their close relation to the enzymes ScELO1, ScELO2, and ScELO3 from Saccharomyces cerevisiae (17Toke D.A. Martin C.E. Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae.J. Biol. Chem. 1996; 271: 18413-18422Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 18Oh C-S. Toke D.A. Mandala S. Martin C.E. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation.J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar), they are thought to code for β-ketoacyl-CoA synthase activities and are often referred to as "elongases," although biochemical data supporting the actual condensing activity are still missing. Yeast and animal cells were used for the expression of most of the elongases catalyzing the elongation steps shown in Fig. 1. Most of the sequences characterized to date are of mammalian origin, but few of them have been studied in sufficient detail to answer all questions regarding regioselectivity and chain length selectivity. Therefore, it remains unclear which substrates other than those indicated in Fig. 1 could also be accepted by these enzymes. Among the few sequences of nonmammalian origin studied in more detail, none was shown to be specific for the elongation of EPA. The Δ5-elongases cloned to date were all isolated from mammals and rather unspecific, in that they carried out multiple elongation reactions not restricted to C20-PUFAs, as shown in Fig. 1. As mentioned above, the synthesis of LCPUFAs in mammals depends on the dietary supply of LA and ALA, and ω6- and ω3-LCPUFAs are each precursors of antagonistic eicosanoid effectors. Typical Western diets are characterized by very high ratios of LA/ALA that are far above the recommended value of ∼5 and thus favor the synthesis of ARA at the expense of EPA and DHA (19Gerster H. Can adults adequately convert α-linolenic acid (18:3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3)?.Int. J. Vit. Nutr. Res. 1998; 68: 159-173PubMed Google Scholar). In addition, because ALA appears to be rapidly degraded by β-oxidation, it seems best to include an appropriate mixture of LCPUFAs in the diet. As this growing demand cannot be met by farmed or landed fish (20Pauly D. Christensen V. Guénette S. Pitcher T.J. Sumaila U.R. Walters C.J. Watson R. Zeller D. Towards sustainability in world fisheries.Nature. 2002; 418: 689-695Crossref PubMed Scopus (2088) Google Scholar) and none of the oilseeds produces LCPUFAs, the implementation of LCPUFA biosynthesis into oilseed crops by modern biotechnology would provide a truly sustainable source of these valuable fatty acids. Because the biosynthesis of DHA according to the Sprecher pathway is clearly too complicated to be reconstituted by gene technology, the alternative route relying on the use of a Δ5-elongase and a Δ4-desaturase is most promising. Whereas all of the desaturases, including the Δ4-desaturase, and the Δ6-elongase have already been isolated from various organisms, an elongase specific for the conversion of a C20-PUFA to a C22-PUFA has not been cloned yet. To reconstitute the simpler pathway of DHA biosynthesis, we started experiments to clone a specific C20-PUFA-elongase restricted in its action to a single elongation cycle to produce a C22-PUFA. Such an activity may become particularly relevant in transgenic plants, in which other potential substrates may be present in excess, leading to complicated mixtures of elongated products. To identify novel elongases, we used the Δ6-elongase sequence from P. patens (PpPSE1) as the query in a tBLASTn (21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70762) Google Scholar) search and identified putative elongase expressed sequence tag (EST) clones from Xenopus laevis (GenBank accession number BC044967), Ciona intestinalis (GenBank accession number AK112719), and Oncorhynchus mykiss (GenBank accession number CA350786). The C. intestinalis clone was kindly provided by S. Fujiwara (22Fujiwara S. Maeda Y. Shin-I T. Kohara Y. Takatori N. Satou Y. Satoh N. Gene expression profiles in Ciona intestinalis cleavage-stage embryos.Mech. Dev. 2002; 112: 115-127Crossref PubMed Scopus (67) Google Scholar), the clone from O. mykiss was a gift from C. E. Rexroad (United States Department of Agriculture, Agricultural Research Service, National Center for Cool and Cold Water Aquaculture), and the X. laevis clone was purchased from the American Type Culture Collection (ATCC 6844054). The open reading frames (ORFs) were amplified by PCR (primers XlELO, CiELO, and OmELO; Table 1) and cloned into the yeast expression vector pYES2.1/V5-His-TOPO (XlELO and CiELO) or pYES3CT (OmELO) from Invitrogen, yielding pXlELO, pCiELO, and pOmELO.TABLE 1PCR primers used in this workGene/ClonePrimerOligonucleotide SequenceXlELOXlELO-5′5′-AGGATCCATGGCCTTCAAGGAGCTCACATC-3′XlELO-3′5′-CCTCGAGTCAATGGTTTTTGCTTTTCAATGCACCG-3′CiELOCiELO-5′5′-TAAGCTTATGGACGTACTTCATCGT-3′CiELO-3′5′-TCAGATCTTTAATCGGTTTTACCATT-3′OmELOOmELO-5′5′-AAGCTTACATAATGGAGACTTTTAA-3′OmELO-3′5′-GGATCCTTCAGTCCCCCCTCACTTCC-3′OtELO1OtELO1-5′5′-CCAAGCTTACATAATGAGTGGCTTACGTGCACCCAA-3′OtELO1-3′5′-CCCTCGAGTCACTGCTGTTTTTTCTGGAGCTTA-3′OtELO2OtELO2-5′5′-CCAAGCTTACATAATGAGCGCCTCCGGTGCGCT-3′OtELO2-3′5′-CCCTCGAGTTAGTCAATTTTTCGAGATCGCGTG-3′PQI119277Tp119GENOMIC-5′5′-ATTGGCGTAATTCTTCGGGG-3′Tp119GENOMIC-3′5′-GTGCTTGTCAAAGTAGAATAAG-3′PQI68798Tp687GENOMIC-5′5′-TCAACCCTCAATACAAAGC-3′Tp687GENOMIC-3′5′-GTGGATGGAAGCTGTTAA-3′TpELO1Tp687ELO-5′5′-ATGGACGCTTACAACGCTGCTATGGACAAGATTGGTGCTGCTATTATTGACTGGTCTGATCCCGATGGAAAGTTCCGTGCCGATAGAGAGGACTGGTGGCTCTGCGACTTCCGT-3′Tp687ELO-3′5′-CTAAGCACTCTTCTTCTTTTTGGGTGC-3′TpELO2Tp119EI-5′5′-ATGTGTTCTCCACCACCATCTCAATCCAAGACTACCTCCTTGTTGGCTAGATACACCACCGCCGCCCTCCTCCTCCTCACCCTCACAACGTGGTGCCACTTCGCCTTCCCAGCCGCC-3′Tp119EI-3′5′-CGTGTGGTGGTAGATGTGGAGGAAGGAGACCTGGTCCATTTTCCCCCTCAACAC-3′Tp119EII-5′5′-TTTATGGTGTTGAGGGGGAAAATGGACCAGGTCTCCTTCCTCCACATCTACCACCACACG-3′Tp119EII-3′5′-CTACATGGCACCAGTAACACG-3′ Open table in a new tab A bacterial artificial chromosome library of Ostreococcus tauri OTHH0595 was constructed and sequenced as previously described (23Derelle E. Ferraz C. Lagoda P. Eychenié S. Cooke R. Regad F. Sabau X. Courties C. Delseny M. Demaille J. Picard A. Moreau H. DNA libraries for sequencing the genome of Ostreococcus tauri (Chlorophytae, Prasinophyceae): the smallest free living eukaryotic cell.J. Phycol. 2002; 38: 1150-1156Crossref Scopus (41) Google Scholar) and used for tBLASTn with PpPSE1 as the query. Two ORFs showing significant similarity to the elongase and apparently lacking introns were amplified by PCR with specific primers (OtELO1 and OtELO2). Fresh cells that were grown in K medium (Sigma-Aldrich) at 23°C in the light and frozen once were used as template. The PCR products were cloned into the pYES2.1/V5-His-TOPO yeast expression vector, yielding pOtELO1 and pOtELO2, respectively. PpPSE1 was also used to blast a genomic library of Thalassiosira pseudonana (Department of Energy Joint Genome Institute), yielding two putative elongase clones (PQI68798 and PQI119277). T. pseudonana SAG 1020-1b (Sammlung für Algenkulturen, Göttingen, Germany) was grown in the light at 23°C in f/2 medium, and the genomic DNA was isolated using the DNeasy Kit (Qiagen). The genomic DNA was used for PCR (primers Tp687GENOMIC and Tp119GENOMIC), and the PCR products were cloned into pGEM-T (Promega) and sequenced. GENESCAN (Arabidopsis algorithm) identified in each clone two exons separated by one intron. The ORF of PQI68798 was amplified by PCR, whereby the 5′ primer was constructed in such a way that it was complementary to exon I (91 bp) and to the 5′ end of exon II (23 bp); therefore, it was missing the part coding for the 142 bp intron (primer Tp687ELO). The 5′ primer of exon I was altered to approximate optimal yeast codon usage without changing the translated sequence. The putative ORF of PQI119277 was also constructed by PCR, taking into account the yeast codon usage. In a first step, both exons were amplified with primers (Tp119EI and Tp119EII) that created a 54 bp overlapping sequence. The overlapping products subsequently served as templates for the second PCR using the 5′ primer of exon I and the 3′ primer of exon II. The ORFs of PQI68798 and PQI119277 were cloned into pYES2.1/V5-His-TOPO, yielding pTpELO1 and pTpELO2, respectively. pXlELO, pCiELO, pOmELO, pOtELO1, pOtELO2, pTpELO1, and pTpELO2 were then used for the transformation of S. cerevisiae 334 (24Hovland P. Flick J. Johnston M. Sclafani R.A. Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose.Gene. 1989; 83: 57-64Crossref PubMed Scopus (142) Google Scholar) or INVSc1 (Invitrogen). For functional expression of the elongases, precultures were grown at 30°C in minimal medium with 2% raffinose lacking the respective amino acid or base for vector selection. Five milliliters of the medium were inoculated with precultures (2 days old) to an optical density at 600 nm of 0.05, and expression was induced with 2% galactose. Expressions were carried out for 4 days at 20°C in the presence of exogenously supplied fatty acids of commercial origin (250–500 μM). Pinolenic acid (18:3Δ5,9,12) was part of a fatty acid mixture isolated from Larix decidua seeds (25Wolff R.L. Lavialle O. Pedrono F. Pasquier E. Deluc L.G. Marpeau A.M. Aitzetmüller K. Fatty acid composition of Pinaceae as taxonomic markers.Lipids. 2001; 36: 439-451Crossref PubMed Scopus (63) Google Scholar). For the coexpression of elongases and desaturases, the yeasts were additionally transformed with the Δ5-desaturase from Phaeodactylum tricornutum (26Domergue F. Lerchl J. Zähringer U. Heinz E. Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis.Eur. J. Biochem. 2002; 269: 4105-4113Crossref PubMed Scopus (135) Google Scholar) and the Δ4-desaturase from Euglena gracilis (11Meyer A. Cirpus P. Ott C. Schlecker R. Zähringer U. Heinz E. Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Δ4-fatty acyl group desaturase.Biochemistry. 2003; 42: 9779-9788Crossref PubMed Scopus (74) Google Scholar). Yeasts harboring the empty vectors (pYES2, pYES3CT, and pESC-Leu) were used as controls. Yeast cells were sedimented by centrifugation and directly used for transmethylation of fatty acids. Fatty acid methyl esters were routinely analyzed by gas liquid chromatography as described previously (11Meyer A. Cirpus P. Ott C. Schlecker R. Zähringer U. Heinz E. Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Δ4-fatty acyl group desaturase.Biochemistry. 2003; 42: 9779-9788Crossref PubMed Scopus (74) Google Scholar), whereas detailed structural identities of new fatty acids were determined by GC-MS (27Sperling P. Lee M. Girke T. Zähringer U. Stymne S. Heinz E. A bifunctional Δ6-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus. A new member of the cytochrome b5 superfamily.Eur. J. Biochem. 2000; 267: 3801-3811Crossref PubMed Scopus (73) Google Scholar). The majority of elongase sequences available in databases originate from mammals (i.e., human, mouse, and rat), and among those functionally characterized, none was shown to be specific for the elongation of Δ6-C18- or Δ5-C20-PUFAs. Because the PpPSE1 gene from the moss P. patens is known to code for a specific Δ6-elongase, we decided to use its translated sequence as the query in a tBLASTn search to identify PUFA-elongases in EST and genomic databases of nonmammalian organisms. The fish O. mykiss, the frog X. laevis, the sea squirt C. intestinalis, and the two DHA-producing algae O. tauri and T. pseudonana were selected as candidate organisms. Among several identified ORFs of interest, we could amplify seven putative elongase clones. The actual numbers of amino acids (272–358) representing the various ORFs as well as their identity compared with the other elongases cloned in the present study are summarized in Table 2. Most of the proteins were only 19–26% identical to PpPSE1, with the exception of OtELO1, which showed a significantly higher identity (42%). Interestingly, the identity between OtELO1 and OtELO2 is only 20%. It should be mentioned that TpELO1 and TpELO2 from T. pseudonana were constructed by PCR after a GENESCAN analysis to delete putative introns and, therefore, may not represent the ORFs translated in the alga. The deduced amino acid sequences of the newly cloned proteins all contained seven to nine putative transmembrane helices as well as the various motifs (16Leonard A.E. Pereira S.L. Sprecher H. Huang Y-S. Elongation of long-chain fatty acids.Prog. Lipid Res. 2004; 43: 36-54Crossref PubMed Scopus (431) Google Scholar) that are typical for this group of elongases (KxxE/DxxDT, the extended histidine box QxxFLHxYHH, the tyrosine box NxxxHxxMYxYY, and TxxQxxQ) (Fig. 2). Lysine residues close to the C terminus that may function as endoplasmic reticulum retention signals were clearly seen in three sequences (XlELO, OtELO1, and TpELO1). The phylogenetic alignment of the currently cloned elongases together with previously characterized enzymes will be discussed below.TABLE 2Elongase genes cloned in this workOrganismSourceOpen Reading FrameAccession NumberPeptide Length (Amino Acids)Identity with PpPSE1Other IdentitiesXenopus laevisExpressed sequence tagXlELOAY60509830225%39% CiELO, 32% OmELO, 24% OtELO2Ciona intestinalisExpressed sequence tagCiELOAY60509928926%36% OmELO, 27% OtELO1, 24% OtELO2Oncorhynchus mykissExpressed sequence tagOmELOAY60510029524%22% OtELO2, 22% OtELO1, 21% TpELO2Ostreococcus tauriGenomicOtELO1AY59133529242%23% TpELO1, 20% OtELO2, 20% TpELO2Ostreococcus tauriGenomicOtELO2AY59133630021%24% XlELO, 23% TpELO2, 20% TpELO1Thalassiosira pseudonanaGenomicTpELO1AY59133727223%22% CiELO, 20% OmELO, 20% XlELOThalassiosira pseudonanaGenomicTpELO2AY59133835819%20% CiELO, 20% XlELO, 17% TpELO1 Open table in a new tab The functions of the proteins encoded by the isolated genes were verified by expression studies in S. cerevisiae comprising incubations with exogenous fatty acids followed by GC-MS analysis of total fatty acid methyl esters. To identify which ORF could encode a Δ5-C20-specific elongase, each ORF was expressed in the presence of either Δ6- or Δ5-polyunsaturated fatty acids (STA or EPA, respectively). Low basal elongation of STA, but no elongation of EPA, was obtained with yeast transformed with the empty vectors (data not shown). All cloned elongases were active in yeast and produced novel fatty acids whose structures were confirmed by GC-MS. Using either STA or EPA as exogenously supplied substrates, the seven elongases could be separated into three different groups: Δ6-C18-PUFA-elongases (TpELO1 and OtELO1), Δ5-C20-PUFA-elongases (TpELO2 and OtELO2), and bifunctional enzymes accepting both C18- and C20-PUFAs (OmELO, XlELO, and CiELO). Results obtained with one member of each group are given in Fig. 3. On expression of TpELO1 (and of OtELO1; data not shown, but see Fig. 4), STA was very efficiently converted to 20:4ω3, whereas EPA was not elongated. In contrast, when OtELO2 (and TpELO2; data not shown, but see Fig. 4) was expressed, EPA was very efficiently elongated, whereas STA elongation did not exceed that of the control strain. When OmELO was expressed, STA was successively elongated to 20:4ω3 and 22:4ω3, whereas EPA was elongated to 22:5ω3. Yeast expressing XlELO or CiELO were also able to convert both STA and EPA (data not shown, but see Fig. 4). As shown in Fig. 3, OmELO was in addition active on 16:1Δ9, 18:1Δ9, and 18:1Δ11, as indicated by the increase/presence of 18:1Δ11, 20:1Δ11, and 20:1Δ13. Starting from 16:1Δ9, OmELO was in fact capable of five successive elongation steps leading to the synthesis of 26:1Δ19.Fig. 4Specificities of functionally expressed elongase enzymes. All transformed yeasts were separately incubated with 250–500 μM of the C18, C20, and C22 fatty acids listed on the abscissa for 4 days, except for pinolenic acid [18:3Δ5,9,12 (18:3Pi)], which was part of a fatty acid mixture isolated from Larix seeds. Fatty acid profiles were analyzed by gas-liquid chromatography. The elongation is given as [product/(substrate + product) × 100]. Each value is the mean ± SD from three to five independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then investigated to what extent these regioselective specificities persisted when fatty acid substrates of different structures, regarding both chain length and position of double bonds, were supplied. The results (Fig. 4) show that TpELO1 and OtELO1 were exclusively active with C18-PUFAs. Although the activity of TpELO1 was restricted to the Δ6-unsaturated fatty acids γ-linolenic acid (GLA) and STA, OtELO1 elongated GLA and STA very efficiently but also to a minor extent Δ9-C18-PUFAs (LA and ALA) and a Δ5-C18-PUFA (pinolenic acid). The activity of the Δ5-C20-PUFA-elong
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