Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesiss⃞s⃞ The online version of this article (available at http://www.jlr.org) contains an additional figure. Published, JLR Papers in Press, August 1, 2004. DOI 10.1194/jlr.M400181-JLR200

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.

[1]  C. J. Lewis,et al.  Identification and expression of mammalian long-chain PUFA elongation enzymes , 2002, Lipids.

[2]  O. Lavialle,et al.  Fatty acid composition of pinaceae as taxonomic markers , 2001, Lipids.

[3]  D. Toke,et al.  ELO2 and ELO3, Homologues of theSaccharomyces cerevisiae ELO1 Gene, Function in Fatty Acid Elongation and Are Required for Sphingolipid Formation* , 1997, The Journal of Biological Chemistry.

[4]  T. Zank,et al.  The evolution of desaturases. , 2003, Prostaglandins, leukotrienes, and essential fatty acids.

[5]  P. Shewry,et al.  Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Hervé Moreau,et al.  DNA LIBRARIES FOR SEQUENCING THE GENOME OF OSTREOCOCCUS TAURI (CHLOROPHYTA, PRASINOPHYCEAE): THE SMALLEST FREE‐LIVING EUKARYOTIC CELL 1 , 2002 .

[7]  S. Nwaka,et al.  Activity of human Δ5 and Δ6 desaturases on multiple n‐3 and n‐6 polyunsaturated fatty acids , 2001 .

[8]  A. Beynen,et al.  The relationships between potassium intakes, transmural potential difference of the rumen epithelium and magnesium absorption in wethers. , 2004, The British journal of nutrition.

[9]  R. J. Henderson,et al.  Biosynthesis of docosahexaenoic acid in trout hepatocytes proceeds via 24-carbon intermediates. , 1997, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[10]  K. Riedel,et al.  Transgenic oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty acids , 2001 .

[11]  D. Toke,et al.  Isolation and Characterization of a Gene Affecting Fatty Acid Elongation in Saccharomyces cerevisiae* , 1996, The Journal of Biological Chemistry.

[12]  R. Reski,et al.  Cloning and functional characterisation of an enzyme involved in the elongation of Delta6-polyunsaturated fatty acids from the moss Physcomitrella patens. , 2002, The Plant journal : for cell and molecular biology.

[13]  M. Crawford,et al.  Is docosahexaenoic acid (DHA) essential? Lessons from DHA status regulation, our ancient diet, epidemiology and randomized controlled trials. , 2004, The Journal of nutrition.

[14]  P. Tvrdik,et al.  Role of a New Mammalian Gene Family in the Biosynthesis of Very Long Chain Fatty Acids and Sphingolipids , 2000, The Journal of cell biology.

[15]  T. Tonon,et al.  Identification of a very long chain polyunsaturated fatty acid Δ4‐desaturase from the microalga Pavlova lutheri 1 , 2003, FEBS letters.

[16]  H. Sprecher,et al.  Elongation of long-chain fatty acids. , 2004, Progress in lipid research.

[17]  Petra Cirpus,et al.  Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase. , 2003, Biochemistry.

[18]  P. Lagali,et al.  Evolutionarily conserved ELOVL4 gene expression in the vertebrate retina. , 2003, Investigative ophthalmology & visual science.

[19]  H. Sprecher.  Metabolism of highly unsaturated n-3 and n-6 fatty acids. , 2000, Biochimica et biophysica acta.

[20]  J. Browse,et al.  Production of Polyunsaturated Fatty Acids by Polyketide Synthases in Both Prokaryotes and Eukaryotes , 2001, Science.

[21]  E. Heinz,et al.  Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis. , 2002, European journal of biochemistry.

[22]  T. Pitcher,et al.  Towards sustainability in world fisheries , 2002, Nature.

[23]  X. Qiu,et al.  Identification of a (cid:1) 4 Fatty Acid Desaturase from Thraustochytrium sp. Involved in the Biosynthesis of Docosahexanoic Acid by Heterologous Expression in Saccharomyces cerevisiae and Brassica , 2001 .

[24]  Min Han,et al.  Suppression of the ELO-2 FA elongation activity results in alterations of the fatty acid composition and multiple physiological defects, including abnormal ultradian rhythms, in Caenorhabditis elegans. , 2003, Genetics.

[25]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[26]  J. Napier,et al.  Identification of a cDNA encoding a novel C18‐Δ9 polyunsaturated fatty acid‐specific elongating activity from the docosahexaenoic acid (DHA)‐producing microalga, Isochrysis galbana 1 , 2002, FEBS letters.

[27]  T. Girke,et al.  A bifunctional Δ6‐fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus , 2000 .

[28]  H. Guillou,et al.  The same rat Delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis. , 2002, The Biochemical journal.

[29]  Y. Huang,et al.  Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[30]  H. Gerster.  Can adults adequately convert alpha-linolenic acid (18:3n-3) to eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3)? , 1998, International journal for vitamin and nutrition research. Internationale Zeitschrift fur Vitamin- und Ernahrungsforschung. Journal international de vitaminologie et de nutrition.

[31]  T. Zank,et al.  Acyl Carriers Used as Substrates by the Desaturases and Elongases Involved in Very Long-chain Polyunsaturated Fatty Acids Biosynthesis Reconstituted in Yeast* , 2003, Journal of Biological Chemistry.

[32]  M. Johnston,et al.  Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose. , 1989, Gene.