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Applied Microbiology and Biotechnology

, Volume 84, Issue 4, pp 709–716 | Cite as

Identification of a novel fatty acid elongase with a wide substrate specificity from arachidonic acid-producing fungus Mortierella alpina 1S-4

  • Eiji Sakuradani
  • Masutoshi Nojiri
  • Haruna Suzuki
  • Sakayu Shimizu
Biotechnologically Relevant Enzymes and Proteins

Abstract

The isolation and characterization of a gene (MALCE1) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4 are described. MALCE1 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae, resulting in the accumulation of 18-, 19-, and 20-carbon monounsaturated fatty acids and eicosanoic acid. Furthermore, the MALCE1 yeast transformant efficiently elongated exogenous 9-hexadecenoic acid, 9,12-octadecadienoic acid, and 9,12,15-octadecatrienoic acid. The MALCE1 gene-silenced strain obtained from M. alpina 1S-4 exhibited a low content of octadecanoic acid and a high content of hexadecanoic acid, compared with those in the wild strain. The enzyme encoded by MALCE1 was demonstrated to be involved in the conversion of hexadecanoic acid to octadecanoic acid, its main role in M. alpina 1S-4.

Keywords

Mortierella alpina Fatty acid elongase RNA interference Gene expression Polyunsaturated fatty acid 

Notes

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research (No. 15658024, to S. S.) from the Ministry of Education, Science, Sports, and Culture, Japan, by the COE for Microbial-Process Development Pioneering Future Production Systems (COE program of the Ministry of Education, Science, Sports, and Culture, Japan to S.S.), and by the Project of the New Energy and Industrial Technology Development Organization, Japan (grant 05A07003d, to E.S.).

References

  1. Ashikari T, Kiuchi-Goto N, Tanaka Y, Shibano Y, Adachi T, Yoshizumi H (1989) High expression and efficient secretion of Rhizopus oryzae glucoamylase in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 30:515–520CrossRefGoogle Scholar
  2. Becker DM, Lundblad V (1994) Manipulation of yeast genes. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. Wiley, New York, pp 13.7.1–13.7.10Google Scholar
  3. Fritzler JM, Millership JJ, Zhu G (2007) Cryptosporidium parvum long-chain fatty acid elongase. Eukaryot Cell 6:2018–2028CrossRefGoogle Scholar
  4. Inagaki K, Aki T, Fukuda Y, Kawamoto S, Shigeta S, Ono K, Suzuki O (2002) Identification and expression of a rat fatty acid elongase involved in the biosynthesis of C18 fatty acids. Biosci Biotechnol Biochem 66:613–621CrossRefGoogle Scholar
  5. Leonard AE, Pereira SL, Sprecher H, Huang YS (2004) Elongation of long-chain fatty acids. Prog Lipid Res 43:36–54CrossRefGoogle Scholar
  6. Oh CS, Toke DA, Mandala S, Martin CE (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 272:17376–17384CrossRefGoogle Scholar
  7. Parker-Barnes JM, Das T, Bobik E, Leonard AE, Thurmond JM, Chaung LT, Huang YS, 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 U S A 97:8284–8289CrossRefGoogle Scholar
  8. Paul S, Gable K, Beaudoin F, Cahoon E, Jaworski J, Napier JA, Dunn TM (2006) Members of the Arabidopsis FAE1-like 3-ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae. J Biol Chem 281:9018–9029CrossRefGoogle Scholar
  9. Prasitchoke P, Kaneko Y, Bamba T, Fukusaki E, Kobayashi A, Harashima S (2007a) Identification and characterization of a very long-chain fatty acid elongase gene in the methylotrophic yeast, Hansenula polymorpha. Gene 391:16–25CrossRefGoogle Scholar
  10. Prasitchoke P, Kaneko Y, Sugiyama M, Bamba T, Fukusaki E, Kobayashi A, Harashima S (2007b) Functional analysis of very long-chain fatty acid elongase gene, HpELO2, in the methylotrophic yeast Hansenula polymorpha. Appl Microbiol Biotechnol 76:417–427CrossRefGoogle Scholar
  11. Qi B, Beaudoin F, Fraser T, Stobart AK, Napier JA, Lazarus CM (2002) 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 510:159–165CrossRefGoogle Scholar
  12. Sakuradani E, Shimizu S (2003) Gene cloning and functional analysis of a second Δ6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Biosci Biotechnol Biochem 67:704–711CrossRefGoogle Scholar
  13. Sakuradani E, Kobayashi M, Shimizu S (1999) Δ9-Fatty acid desaturase from arachidonic acid-producing fungus. Unique gene sequence and its heterologous expression in a fungus, Aspergillus. Eur J Biochem 260:208–216CrossRefGoogle Scholar
  14. Sakuradani E, Murata S, Kanamaru H, Shimizu S (2008) Functional analysis of a fatty acid elongase from arachidonic acid-producing Mortierella alpina 1S-4. Appl Microbiol Biotechnol 81:497–503CrossRefGoogle Scholar
  15. Takeno S, Sakuradani E, Murata S, Inohara-Ochiai M, Kawashima H, Ashikari T, Shimizu S (2004) Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4. Appl Microbiol Biotechnol 65:419–425CrossRefGoogle Scholar
  16. Takeno S, Sakuradani E, Murata S, Inohara-Ochiai M, Kawashima H, Ashikari T, Shimizu S (2005a) Molecular evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of an elongase. Lipids 40:25–30CrossRefGoogle Scholar
  17. Takeno S, Sakuradani E, Tomi A, Inohara-Ochiai M, Kawashima H, Ashikari T, Shimizu S (2005b) Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with Δ12-desaturase gene expression. Appl Environ Microbiol 71:5124–5128CrossRefGoogle Scholar
  18. Toke DA, Martin CE (1996) Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. J Biol Chem 271:18413–18422CrossRefGoogle Scholar
  19. Yamada H, Shimizu S, Shinmen Y (1987) Production of arachidonic acid by Mortierella elongata 1S-5. Agric Biol Chem 51:785–790Google Scholar
  20. Zhang S, Sakuradani E, Ito K, Shimizu S (2007) Identification of a novel bifunctional Δ12/Δ15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2. FEBS Lett 581:315–319CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Eiji Sakuradani
    • 1
  • Masutoshi Nojiri
    • 1
  • Haruna Suzuki
    • 1
  • Sakayu Shimizu
    • 1
  1. 1.Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversityKyotoJapan

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