1887

Abstract

A third gene (Δ9-3) encoding a fatty acid Δ9-desaturase was isolated from the oil-producing fungus . The predicted protein of 512 aa shared 53% sequence identity with the two fatty acid Δ9-desaturases, ole1p and ole2p, already described in this organism and contained three histidine boxes, four putative transmembrane domains and a C-terminal cytochrome fusion that are typical of most fungal membrane-bound fatty acid desaturases. However, unlike the and genes, the Δ9-3 ORF failed to complement the mutation. GC-MS analysis of fatty-acid-supplemented yeast transformants containing the Δ9-3 gene indicated that this enzyme had negligible activity with endogenous palmitic acid (16:0) as substrate and moderate activity (30–65% desaturation) with endogenous stearic acid (18:0). Yeast transformants overexpressing any one of the three fatty acid Δ9-desaturase genes or the gene produced low amounts of hexacosenoic acid [26:1(n-9)], a fatty acid that is not normally present in yeast cells. It follows that these Δ9-desaturases may also display low n-9 desaturation activity with very long-chain saturated fatty acid substrates. Conversely, high levels of desaturase in the endoplasmic reticulum membrane of these yeast transformants may increase the availability of suitable monounsaturated substrates for fatty acid elongation.

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2002-06-01
2024-03-29
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References

  1. Certik M., Sakuradani E., Shimizu S. 1998; Desaturase-defective fungal mutants: useful tools for the regulation and overproduction of polyunsaturated fatty acids. Trends Biotechnol 16:500–505 [CrossRef]
    [Google Scholar]
  2. Elble R. 1992; A simple and efficient procedure for transformation of yeasts. BioTechniques 13:18–20
    [Google Scholar]
  3. Fay L., Richli U. 1991; Location of double bonds in polyunsaturated fatty acids by gas chromatography-mass spectrometry after 4,4-dimethyloxazoline derivatization. J Chromatogr 541:89–98 [CrossRef]
    [Google Scholar]
  4. Fukuchi-Mizutani M., Tasaka Y., Tanaka Y., Ashikari T., Kusumi T., Murata N. 1998; Characterization of Δ9 acyl-lipid desaturase homologues from Arabidopsis thaliana . Plant Cell Physiol 39:247–253 [CrossRef]
    [Google Scholar]
  5. Gargano S., Di Lallo G., Kobayashi G. S., Maresca B. 1995; A temperature-sensitive strain of Histoplasma capsulatum has an altered Δ9-fatty acid desaturase gene. Lipids 30:899–906 [CrossRef]
    [Google Scholar]
  6. Gietz R. D., Schiestl R. H., Willems A. R., Woods R. A. 1995; Studies on the transformation of intact yeast cells by the LiAc/ss-DNA/PEG procedure. Yeast 11:355–360 [CrossRef]
    [Google Scholar]
  7. Gonzalez C. I., Martin C. E. 1996; Fatty acid-responsive control of mRNA stability. J Biol Chem 271:25801–25809 [CrossRef]
    [Google Scholar]
  8. Gurr S. J., Unkles S. E., Kinghorn J. R. 1987; The structure and organization of nuclear genes of filamentous fungi. In Gene Structure in Eukaryotic Microbes pp 93–139 Edited by Kinghorn J. R. Oxford: IRL Press;
    [Google Scholar]
  9. Huang Y.-S., Chaudhary S., Thurmond J. M., Bobik E. G. Jr, Yuan L., Chan G. M., Kirchner S. J., Mukerji P., Knutzon D. S. 1999; Cloning of Δ12- and Δ6-desaturases from Mortierella alpina and recombinant production of γ-linolenic acid in Saccharomyces cerevisiae . Lipids 34:649–659 [CrossRef]
    [Google Scholar]
  10. Jackson F. M., Fraser T. C. M., Smith M. A., Lazarus C., Stobart A. K., Griffiths G. 1998; Biosynthesis of C18 polyunsaturated fatty acids in microsomal membrane preparations from the filamentous fungus Mucor circinelloides . Eur J Biochem 252:513–519 [CrossRef]
    [Google Scholar]
  11. Kobayashi M., Sakuradani E., Shimizu S. 1999; Genetic analysis of cytochrome b 5 from arachidonic acid-producing fungus, Mortierella alpina 1S-4: cloning, RNA editing and expression of the gene in Escherichia coli , and purification and characterisation of the gene product. J Biochem 125:1094–1103 [CrossRef]
    [Google Scholar]
  12. Kohlwein S. D., Eder S., Oh C.-S., Martin C. E., Gable K., Bacikova D., Dunn T. 2001; Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear–vacuolar interface in Saccharomyces cerevisiae . Mol Cell Biol 21:109–125 [CrossRef]
    [Google Scholar]
  13. Libisch B., Michaelson L. V., Lewis M. J., Shewry P. R., Napier J. A. 2000; Chimeras of Δ6-fatty acid and Δ8-sphingolipid desaturases. Biochem Biophys Res Commun 279:779–785 [CrossRef]
    [Google Scholar]
  14. MacKenzie D. A., Wongwathanarat P., Carter A. T., Archer D. B. 2000; Isolation and use of a homologous histone H4 promoter and a ribosomal DNA region in a transformation vector for the oil-producing fungus Mortierella alpina . Appl Environ Microbiol 66:4655–4661 [CrossRef]
    [Google Scholar]
  15. McDonough V. M., Stukey J. E., Martin C. E. 1992; Specificity of unsaturated fatty acid-regulated expression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem 267:5931–5936
    [Google Scholar]
  16. Meesters P. A. E. P., Eggink G. 1996; Isolation and characterization of a Δ9-fatty acid desaturase gene from the oleaginous yeast Cryptococcus curvatus CBS 570. Yeast 12:723–730 [CrossRef]
    [Google Scholar]
  17. Michaelson L. V., Lazarus C. M., Griffiths G., Napier J. A., Stobart A. K. 1998; Isolation of a Δ5-fatty acid desaturase gene from Mortierella alpina . J Biol Chem 273:19055–19059 [CrossRef]
    [Google Scholar]
  18. Napier J. A., Sayanova O., Sperling P., Heinz E. 1999; A growing family of cytochrome b 5-domain fusion proteins. Trends Plant Sci 4:2–4 [CrossRef]
    [Google Scholar]
  19. Oh C.-S., Toke D. A., Mandala S., Martin C. E. 1997; ELO2 and ELO3 , homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation. J Biol Chem 272:17376–17384 [CrossRef]
    [Google Scholar]
  20. Page R. D. M. 1996; treeview: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358
    [Google Scholar]
  21. Parker-Barnes J. M., Das T., Bobik E., Leonard A. E., Thurmond J. M., Chaung L.-T., Huang Y.-S., Mukerji P. 2000; Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids. Proc Natl Acad Sci USA 97:8284–8289 [CrossRef]
    [Google Scholar]
  22. Ratledge C. 1993; Single cell oils – have they a biotechnological future?. Trends Biotechnol 11:278–284 [CrossRef]
    [Google Scholar]
  23. Sakuradani E., Kobayashi M., Shimizu S. 1999; Δ6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus – gene cloning and its heterologous expression in a fungus, Aspergillus . Gene 238:445–453 [CrossRef]
    [Google Scholar]
  24. Sedgwick S. G., Smerdon S. J. 1999; The ankyrin repeat: a diversity of interactions on a common structural framework. Trends Biotechnol 24:311–316
    [Google Scholar]
  25. Sperling P., Heinz E. 2001; Desaturases fused to their electron donor. Eur J Lipid Sci Technol 103:158–180 [CrossRef]
    [Google Scholar]
  26. Sperling P., Zähringer U., Heinz E. 1998; A sphingolipid desaturase from higher plants. J Biol Chem 273:28590–28596 [CrossRef]
    [Google Scholar]
  27. Stukey J. E., McDonough V. M., Martin C. E. 1989; Isolation and characterization of OLE1 , a gene affecting fatty acid desaturation from Saccharomyces cerevisiae . J Biol Chem 264:16537–16544
    [Google Scholar]
  28. Stukey J. E., McDonough V. M., Martin C. E. 1990; The OLE1 gene of Saccharomyces cerevisiae encodes the Δ9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene. J Biol Chem 265:20144–20149
    [Google Scholar]
  29. Tabor D. E., Xia Y.-R., Mehrabian M., Edwards P. A., Lusis A. J. 1998; A cluster of stearoyl CoA desaturase genes, Scd1 and Scd2 , on mouse chromosome 19. Mamm Genome 9:341–342 [CrossRef]
    [Google Scholar]
  30. Thiede M. A., Ozols J., Strittmatter P. 1986; Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase. J Biol Chem 261:13230–13235
    [Google Scholar]
  31. Tiku P. E., Gracey A. Y., Macartney A. I., Beynon R. J., Cossins A. R. 1996; Cold-induced expression of Δ9-desaturase in carp by transcriptional and posttranscriptional mechanisms. Science 271:815–818 [CrossRef]
    [Google Scholar]
  32. Vernet T., Dignard D., Thomas D. Y. 1987; A family of yeast expression vectors containing the phage-f1 intergenic region. Gene 52:225–233 [CrossRef]
    [Google Scholar]
  33. Wach A., Brachat A., Pöhlmann R., Philippsen P. 1994; New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae . Yeast 10:1793–1808 [CrossRef]
    [Google Scholar]
  34. Watts J. L., Browse J. 2000; A palmitoyl-CoA-specific Δ9 fatty acid desaturase from Caenorhabditis elegans . Biochem Biophys Res Commun 272:263–269 [CrossRef]
    [Google Scholar]
  35. Welch J. W., Burlingame A. L. 1973; Very long-chain fatty acids in yeast. J Bacteriol 115:464–466
    [Google Scholar]
  36. Wicker-Thomas C., Henriet C., Dallerac R. 1997; Partial characterization of a fatty acid desaturase gene in Drosophila melanogaster . Insect Biochem Mol Biol 27:963–972 [CrossRef]
    [Google Scholar]
  37. Winzeler E. A., Shoemaker D. D., Astromoff A. 49 other authors 1999; Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906 [CrossRef]
    [Google Scholar]
  38. Wongwathanarat P., Michaelson L. V., Carter A. T., Lazarus C. M., Griffiths G., Stobart A. K., Archer D. B., MacKenzie D. A. 1999; Two fatty acid Δ9-desaturase genes, ole1 and ole2 , from Mortierella alpina complement the yeast ole1 mutation. Microbiology 145:2939–2946
    [Google Scholar]
  39. Yukawa Y., Takaiwa F., Shoji K., Masuda K., Yamada K. 1996; Structure and expression of two seed-specific cDNA clones encoding stearoyl-acyl carrier protein desaturase from sesame, Sesamum indicum L. Plant Cell Physiol 37:201–205 [CrossRef]
    [Google Scholar]
  40. Zheng Y., Prouty S. M., Harmon A., Sundberg J. P., Stenn K. S., Parimoo S. 2001; Scd3 – a novel gene of the stearoyl-CoA desaturase family with restricted expression in skin. Genomics 71:182–191 [CrossRef]
    [Google Scholar]
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