Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Identification and Heterologous Expression of a ${\Delta}4$-Fatty Acid Desaturase Gene from Isochrysis sphaerica

  • Guo, Bing (Oil Crops Research Institute of Chinese Academy of Agricultural Sciences) ;
  • Jiang, Mulan (Oil Crops Research Institute of Chinese Academy of Agricultural Sciences) ;
  • Wan, Xia (Oil Crops Research Institute of Chinese Academy of Agricultural Sciences) ;
  • Gong, Yangmin (Oil Crops Research Institute of Chinese Academy of Agricultural Sciences) ;
  • Liang, Zhuo (Oil Crops Research Institute of Chinese Academy of Agricultural Sciences) ;
  • Hu, Chuanjiong (Oil Crops Research Institute of Chinese Academy of Agricultural Sciences)
  • Received : 2013.01.25
  • Accepted : 2013.06.30
  • Published : 2013.10.28
Download PDF
⟨ Previous Next ⟩

Abstract

The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, $C20:5{\omega}-3$) and docosahexaenoic acid (DHA, $C22:6{\omega}-3$) that are important to human health. Here, we report a functional characterization of a ${\Delta}4$-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops.

Keywords

References

  1. DePauw N, Personne G. 1988. Micro-algal biotechnology, p. 197. In Borowitzka MA, Borowitzka JL (eds.). Microalgal Biotechnology, Cambridge University Press, Cambridge.
  2. El Abed MM, Marzouk B, Medhioub MN, Helal AN, Medhioub A. 2008. Microalgae: a potential source of polyunsaturated fatty acids. Nutr. Health 19: 221-226. https://doi.org/10.1177/026010600801900309
  3. Gil A, Ramirez M, Gi M. 2003. Role of long-chain polyunsaturated fatty acids in infant nutrition. Eur. J. Clin. Nutr. 57: 31-34. https://doi.org/10.1038/sj.ejcn.1601517
  4. Guo B, Gong YM, Wan X, Liang Z, Jiang ML. 2011. The effect of light intensity and temperature on growth and fatty acid composition and contents of Isochrysis sphaerica. Chin. J. Oil Crop Sci. 33: 295-301 [in Chinese].
  5. Hartwich J, Malec MM, Partyka L, Perez-Martinez P, Marin C, Lopez-Miranda J, et al. 2009. The effect of the plasma n-3/n-6 polyunsaturated fatty acid ratio on the dietary LDL phenotype transformation - insights from the LIPGENE study. Clin. Nutr. 28: 510-515. https://doi.org/10.1016/j.clnu.2009.04.016
  6. Huang YS, Chaudhary S, Thurmond JM, Bobik EG, Yuan L, Chan GM, et al. 1999. Cloning of ${\Delta}12$- and ${\Delta}6$-desaturases from Mortierella alpina and recombinant production of ${\gamma}$-linolenic acid in Saccharomyces cerevisiae. Lipids 34: 649-659. https://doi.org/10.1007/s11745-999-0410-8
  7. Jiang Y, Fan KW, Wong RT, Chen F. 2004. Fatty acid composition and squalene content of the marine microalga Schizochytrium mangrovei. J. Agric. Food Chem. 52: 1196-1200. https://doi.org/10.1021/jf035004c
  8. Kajikawa M, Yamato KT, Sakai Y, Fukuzawa H, Ohyama K, Kohchi T. 2006. Isolation and functional characterization of fatty acid delta5-elongase gene from the liverwort Marchantia polymorpha L. FEBS Lett. 580: 149-154. https://doi.org/10.1016/j.febslet.2005.11.065
  9. Kumon Y, Kamisaka Y, Tomita N, Kimura K, Uemura H, Yokochi T, et al. 2008. Isolation and characterization of a delta5-desaturase from Oblongichytrium sp. Biosci. Biotechnol. Biochem. 72: 2224-2227. https://doi.org/10.1271/bbb.80235
  10. Kyle DJ, Arterburn LM. 1998. Single cell oil sources of docosahexaenoic acid: clinical studies. World Rev. Nutr. Diet. 83: 116-131. https://doi.org/10.1159/000059656
  11. Lee WH, Cho KW, Park SY, Shin KS, Lee DS, Hwang SK, et al. 2008. Identification of a psychrophile Shewanella sp. KMG427 as an eicosapentaenoic acid producer. J. Microbiol. Biotechnol. 18: 1869-1873.
  12. Liu YG, Whittier RF. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from p1 and YAC clones for chromosome walking. Genomics 25: 674-681. https://doi.org/10.1016/0888-7543(95)80010-J
  13. Liu YG, Chen Y. 2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43: 649-650. https://doi.org/10.2144/000112601
  14. Li YT, Li MT, Fu CH, Zhou PP, Liu JM, Yu LJ. 2009. Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into Pichia pastoris. Biotechnol. Lett. 31: 1011-1017. https://doi.org/10.1007/s10529-009-9970-z
  15. Napier JA, Michaelson LV, Stobart AK. 1999. Plant desaturases: harvesting the fat of the land. Curr. Opin. Plant Biol. 2: 123-127. https://doi.org/10.1016/S1369-5266(99)80025-9
  16. Neuringer M. 2000. Infant vision and retinal function in studies of dietary long-chain polyunsaturated fatty acids: methods, results, and implications. Am. J. Clin. Nutr. 71: 256-267. https://doi.org/10.1093/ajcn/71.1.256S
  17. Novgorodtseva TP, Karaman YK, Zhukova NV, Lobanova EG, Antonyuk MV, Kantur TA. 2011. Composition of fatty acids in plasma and erythrocytes and eicosanoids level in patients with metabolic syndrome. Lipids Health Dis. 10: 82. https://doi.org/10.1186/1476-511X-10-82
  18. Pereira SL, Leonard AE, Huang YS, Chuang LT, Mukerji P. 2004. Identification of two novel microalgal enzymes involved in the conversion of the omega3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid. Biochem. J. 384: 357-366. https://doi.org/10.1042/BJ20040970
  19. Qiu X, Hong H, Mackenzie SL. 2001. Identification of a Delta 4 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. 276: 31564-31566.
  20. Robert SS. 2006. Production of eicosapentaenoic and docosahexaenoic acid-containing oils in transgenic land plants for human and aquaculture nutrition. Mar. Biotechnol. (NY) 8: 103-109. https://doi.org/10.1007/s10126-005-5142-x
  21. Sayanova OV, Napier JA. 2004. Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants. Phytochemistry 65: 147-158. https://doi.org/10.1016/j.phytochem.2003.10.017
  22. Shi T, Yu A, Li M, Ou X, Xing L, Li M. 2012. Identification of a novel C22-4-producing docosahexaenoic acid (DHA) specific polyunsaturated fatty acid desaturase gene from Isochrysis galbana and its expression in Saccharomyces cerevisiae. Biotechnol. Lett. 34: 2265-2274. https://doi.org/10.1007/s10529-012-1028-y
  23. Singer T, Burke E. 2003. High-throughput TAIL-PCR as a tool to identify DNA flanking insertions. Methods Mol. Biol. 236: 241-272.
  24. Tonon T, Harvey D, Larson TR, Graham IA. 2003. Identification of a very long chain polyunsaturated fatty acid delta4-desaturase from the microalga Pavlova lutheri. FEBS Lett. 553: 440-444. https://doi.org/10.1016/S0014-5793(03)01078-0
  25. Tully AM, Roche HM, Doyle R, Fallon C, Bruce I, Lawlor B, et al. 2003. Low serum cholesteryl ester-docosahexaenoic acid levels in Alzheimer's disease: a case-control study. Br. J. Nutr. 89: 483-489. https://doi.org/10.1079/BJN2002804
  26. Zank TK, Zahringer U, Beckmann C, Pohnert G, Boland W, Holtorf H, et al. 2002. Cloning and functional characterisation of an enzyme involved in the elongation of delta6-polyunsaturated fatty acids from the moss Physcomitrella patens. Plant J. 31: 255-268. https://doi.org/10.1046/j.1365-313X.2002.01354.x
  27. Zhang YB, Jiang Q, Jiang ML, Ma LX. 2004. Cloning of a laccase gene from Flammulina velutipes and study on its expression in Pichia pastoris. Wei Sheng Wu Xue Bao 44: 775-779.

Cited by

  1. Isolation and Characterization of the Diatom Phaeodactylum Δ5-Elongase Gene for Transgenic LC-PUFA Production in Pichia pastoris vol.12, pp.3, 2013, https://doi.org/10.3390/md12031317
  2. Effect of cerulenin on fatty acid composition and gene expression pattern of DHA-producing strain Colwellia psychrerythraea strain 34H vol.15, pp.None, 2013, https://doi.org/10.1186/s12934-016-0431-9