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Cloning of Flax Oleic Fatty Acid Desaturase and Its Expression in Yeast

  • Original Paper
  • Published:
Journal of the American Oil Chemists' Society

Abstract

Fatty acid desaturases dehydrogenate acyl chains, which results in the formation of a double bond. Using PCR on flax genomic DNA, we cloned a putative Δ12 fatty acid desaturase (Fad2) gene encoding a 378 amino acid protein. Heterologous expression of this protein in yeast as an N-terminal fusion to GFP showed its localization within endoplasmic reticulum. Analysis of membrane lipids revealed the production of dienoic fatty acids, decreased levels of FAD2 substrates and an increased concentration of longer fatty acids. Higher peroxidation of lipids in FAD2-containing strains is not reflected by any visible phenotype in YPD medium. However, FAD2-containing strains with deleted superoxide dismutase genes exhibited significant growth reductions under oxidative stress.

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Abbreviations

GFP:

Green fluorescent protein

FAD2:

Oleic acid (Δ12) fatty acid desaturase

FA:

Fatty acid

PUFA:

Polyunsaturated fatty acid

TAG:

Triacylglycerols

SOD:

Superoxide dismutase

FAMEs:

Fatty acid methyl esters

References

  1. Buist PH (2004) Fatty acid desaturases: selecting the dehydrogenation channel. Nat Prod Rep 21:249–262

    Article  CAS  Google Scholar 

  2. Sánchez-García A, Mancha M, Heinz E, Martínez-Rivas JM (2004) Differential temperature regulation of three sunflower microsomal oleate desaturase (FAD2) isoforms overexpressed in Saccharomyces cerevisiae. Eur J Lipid Sci Technol 106:583–590

    Article  CAS  Google Scholar 

  3. Martin M, Leon J, Dammann C, Albar JP, Griffiths G, Sanchez-Serrano JJ (1999) Antisense-mediated depletion of potato leaf omega3 fatty acid desaturase lowers linolenic acid content and reduces gene activation in response to wounding. Eur J Biochem 262:283–290

    Article  CAS  Google Scholar 

  4. Kachroo P, Shanklin J, Shah J, Whittle EJ, Klessig DF (2001) A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Proc Natl Acad Sci USA 98:9448–9453

    Article  CAS  Google Scholar 

  5. Ohlrogge J, Browse J (1995) Lipid biosynthesis. Plant Cell 7:957–970

    Article  CAS  Google Scholar 

  6. Mori TA, Beilin LJ (2004) Omega-3 fatty acids and inflammation. Curr Atheroscler Rep 6:461–467

    Article  Google Scholar 

  7. Olson RE (2003) Nutrition and genetics: an expanding frontier. Am J Clin Nutr 78:201–208

    CAS  Google Scholar 

  8. Lukaszewicz M, Szopa J, Krasowska A (2004) Susceptibility of lipids from different flax cultivars to peroxidation and its lowering by added antioxidants. Elsevier, Amsterdam

    Google Scholar 

  9. Liu Q, Singh SP, Green AG (2002) High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol 129:1732–1743

    Article  CAS  Google Scholar 

  10. Buhr T, Sato S, Ebrahim F, Xing A, Zhou Y, Mathiesen M, Schweiger B, Kinney A, Staswick P (2002) Ribozyme termination of RNA transcripts down-regulate seed fatty acid genes in transgenic soybean. Plant J 30:155–163

    Article  CAS  Google Scholar 

  11. Alicia Sánchez-García MM, Heinz E, Martínez-Rivas JM (2004) Differential temperature regulation of three sunflower microsomal oleate desaturase (FAD2) isoforms overexpressed in Saccharomyces cerevisiae. Eur J Lipid Sci Technol 106:583–590

    Article  CAS  Google Scholar 

  12. Heppard EP, Kinney AJ, Stecca KL, Miao GH (1996) Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans. Plant Physiol 110:311–319

    Article  CAS  Google Scholar 

  13. Krasowska A, Dziadkowiec D, Lukaszewicz M, Wojtowicz K, Sigler K (2003) Effect of antioxidants on Saccharomyces cerevisiae mutants deficient in superoxide dismutases. Folia Microbiol (Praha) 48:754–760

    CAS  Google Scholar 

  14. Balzi E, Chen W, Ulaszewski S, Capieaux E, Goffeau A (1987) The multidrug resistance gene PDR1 from Saccharomyces cerevisiae. J Biol Chem 262:16871–16879

    CAS  Google Scholar 

  15. Wach A, Brachat A, Rebischung C, Steiner S, Pokorni K, Heesen S, Philippsen P (1998) PCR-based gene targeting in Saccharomyces cerevisiae. Methods Microbiol 26:67–81

    Article  CAS  Google Scholar 

  16. Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524

    Article  CAS  Google Scholar 

  17. Allen CF, Good P (1971) Acyl lipids in photosynthetic systems. Methods Enzymol 123:523–547

    Article  Google Scholar 

  18. Prescha A, Swiedrych A, Biernat J, Szopa J (2001) Increase in lipid content in potato tubers modified by 14-3-3 gene overexpression. J Agric Food Chem 49:3638–3643

    Article  CAS  Google Scholar 

  19. Lukaszewicz M, Feuermann M, Jerouville B, Stas A, Boutry M (2000) In vivo evaluation of the context sequence of the translation initiation codon in plants. Plant Sci 154:89–98

    Article  CAS  Google Scholar 

  20. Jin UH, Lee JW, Chung YS, Lee JH, Yi YB, Kim YK, Hyung NI, Pyee JH, Chung CH (2001) Characterization and temporal expression of a omega-6 fatty acid desaturase cDNA from sesame (Sesamum indicum L.) seeds. Plant Sci 161:935–941

    Article  CAS  Google Scholar 

  21. Tatzer V, Zellnig G, Kohlwein SD, Schneiter R (2002) Lipid-dependent subcellular relocalization of the acyl chain desaturase in yeast. Mol Biol Cell 13:4429–4442

    Article  CAS  Google Scholar 

  22. Reed DW, Schafer UA, Covello PS (2000) Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae. Plant Physiol 122:715–720

    Article  CAS  Google Scholar 

  23. Pirtle IL, Kongcharoensuntorn W, Nampaisansuk M, Knesek JE, Chapman KD, Pirtle RM (2001) Molecular cloning and functional expression of the gene for a cotton Delta-12 fatty acid desaturase (FAD2). Biochim Biophys Acta 1522:122–129

    CAS  Google Scholar 

  24. Covello PS, Reed DW (1996) Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae. Plant Physiol 111:223–226

    Article  CAS  Google Scholar 

  25. Schwartzbeck JL, Jung S, Abbott AG, Mosley E, Lewis S, Pries GL, Powell GL (2001) Endoplasmic oleoyl-PC desaturase references the second double bond. Phytochemistry 57:643–652

    Article  CAS  Google Scholar 

  26. Heilmann I, Pidkowich MS, Girke T, Shanklin J (2004) Switching desaturase enzyme specificity by alternate subcellular targeting. Proc Natl Acad Sci USA 101:10266–10271

    Article  CAS  Google Scholar 

  27. Broadwater JA, Whittle E, Shanklin J (2002) Desaturation and hydroxylation. Residues 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity. J Biol Chem 277:15613–15620

    Article  CAS  Google Scholar 

  28. Bossie MA, Martin CE (1989) Nutritional regulation of yeast delta-9 fatty acid desaturase activity. J Bacteriol 171:6409–6413

    CAS  Google Scholar 

  29. Okuyama H, Saito M, Joshi VC, Gunsberg S, Wakil SJ (1979) Regulation by temperature of the chain length of fatty acids in yeast. J Biol Chem 254:12281–12284

    CAS  Google Scholar 

  30. Kvam E, Gable K, Dunn TM, Goldfarb DS (2005) Targeting of Tsc13p to nucleus–vacuole junctions: a role for very-long-chain fatty acids in the biogenesis of microautophagic vesicles. Mol Biol Cell 16:3987–3998

    Article  CAS  Google Scholar 

  31. Temple MD, Perrone GG, Dawes IW (2005) Complex cellular responses to reactive oxygen species. Trends Cell Biol 15:319–326

    Article  CAS  Google Scholar 

  32. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:3497–3500

    Article  CAS  Google Scholar 

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Acknowledgments

The authors wish to thank Marc Boutry and Benedict Purnelle for DNA sequencing and J.H. Hegemann for providing the pUG36 vector.

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Correspondence to Marcin Łukaszewicz.

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Krasowska, A., Dziadkowiec, D., Polinceusz, A. et al. Cloning of Flax Oleic Fatty Acid Desaturase and Its Expression in Yeast. J Am Oil Chem Soc 84, 809–816 (2007). https://doi.org/10.1007/s11746-007-1106-9

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  • DOI: https://doi.org/10.1007/s11746-007-1106-9

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