Abstract
In oleaginous fungus Cunninghamella echinulata, Δ9-fatty acid desaturase introduces the first double bond into a saturated fatty acid. Three distinct genes, designated as d9dma, d9dmb and d9dmc, all encoding putative Δ9-fatty acid desaturases were isolated from this strain. The predicted proteins showed 79–87 % identity to other fungal Δ9-fatty acid desaturases. They all contain three conserved histidine boxes, C-terminal cytochrome b 5 fusion and four transmembrane domains characteristic of Δ9-desaturase. Each putative Δ9-desaturase gene from C. echinulata was able to complement the ole1 mutation in Saccharomyces cerevisiae L8-14C through heterologous expression. Analysis of the fatty acid composition of the transgenic yeast revealed that the conversion rates of 16:0 and 18:0 by D9DMA were obviously higher than those of D9DMB and D9DMC. In addition, D9DMA, D9DMB and D9DMC all had a substrate preference for 18:0 compared with 16:0. Of interest, D9DMA could saturate 12:0, 14:0, 16:0, 17:0, 18:0 and 20:0, while D9DMB saturated 14:0, 16:0, 17:0, 18:0 and 20:0. We also noticed that the transcriptional level of d9dma in C. echinulata was stimulated by cell growth but not by decline in temperature. In contrast, expression of d9dmb and d9dmc was regulated by neither cell growth nor decline in temperature in this strain.
Similar content being viewed by others
References
Mansilla MC, Aguilar PS, Albanesi D, Cybulski LE, de Altabe S, Mendoza D (2003) Regulation of fatty acid desaturation in Bacillus subtilis. Prostaglandins Leukot Essent Fatty Acids 2:187–190
Mansilla MC, Banchio CE, de Mendoza D (2008) Signalling pathways controlling fatty acid desaturation. Subcell Biochem 49:71–99
Flowers MT, Ntambi JM (2008) Role of stearoyl-coenzyme A desaturase in regulation lipid metabolism. Curr Opin Lipidol 19:248–256
Sperling P, Schmidt H, Heinz E (1995) A cytochrome-b5-containing fusion protein similar to plant acyl lipid desaturases. Eur J Biochem 232:798–805
Li Y, Xu X, Dietrich M, Urlacher VB, Schmid RD, Ouyang P, He B (2009) Identification and functional expression of a Δ9 fatty acid desaturase from marine bacterium Pseudoalteromonas sp. MLY15. J Mol Catal B Enzyme 56:96–101
Miyazaki M, Bruggink SM, Ntambi JM (2006) Identification of mouse palmitoyl-coenzyme A Δ9-desaturase. J Lipid Res 47:700–704
Sakuradani E, Kobayashi M, Shimizu S (1999) Δ9-fatty acid desaturase from arachidonic acid-producing fungus. Eur J Biochem 260:208–216
Watts JL, Browse J (2000) A palmitoyl-CoA-Specific Δ9 fatty acid desaturase from Caenorhabditis elegans. Biochem Biophys Res Commun 272:263–269
Stukey JE, McDonough VM, Martin CE (1989) Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae. J Biol Chem 264:16537–16544
Kajiwara S (2002) Molecular cloning and characterization of the delta9 fatty acid desaturase gene and its promoter region from Saccharomyces kluyveri. FEMS Yeast Res 2:333–339
Lu SF, Tolstorukov II, Anamnart S, Kaneko Y, Harashima S (2000) Cloning, sequencing, and functional analysis of H-OLE1 gene encoding Δ9 fatty acid desaturase in Hansenula polymorpha. Appl Microbiol Biotechnol 54:499–509
Sakai H, Kajiwara S (2003) A stearoyl-CoA-specific Δ9 fatty acid desaturase from Basidiomycete Lentinula edodes. Biosci Biotechnol Biochem 67:2431–2437
Wei D, Zhou H, Yang Z, Zhang X, Xing L, Li M (2009) Identification of a novel delta 9-fatty acid desaturase gene and its promoter form oil-producing fungus Rhizopus arrhizus. Mol Biol Rep 36:177–186
Wilson RA, Chang PK, Dobrzyn A, Ntambi JM, Zarnowski R, Keller NP (2004) Two Δ9-stearic acid desaturases are required for Aspergillus nidulans growth and development. Fungal Genet Biol 41:501–509
Wongwathanarat P, Michaelson LV, Carter AT, Lazarus CM, Griffiths G, Keith Stobart A, Archer DB, MacKenzie DA (1999) Two fatty acid Δ9-desaturase genes, ole1 and ole2, from Mortierella alpina complement the yeast ole1 mutaion. Microbiology 145:2939–2946
Martin CE, Oh CS, Jiang Y (2007) Regulation of long chain unsaturated fatty acid synthesis in yeast. Biochim Biophys Acta 1771:271–285
MacKenzie DA, Carter AT, Wongwathanarat P, Eagles J, Salt J, Archer DB (2002) A third fatty acid Δ9 fatty acid desaturase from Mortierella alpina with a different substrate specificity to ole1p and ole2p. Microbiology 148:1725–1735
Tiku PE, Gracey AY, Macartney AL, Beynon RJ, Cossins AR (1996) Cold-induced expression of delta 9-desaturase in carp by transcriptional and posttranslational mechanisms. Science 271:815–818
Wiker-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
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
Savory FR, Salt SM, Hop IA (2011) DAF-16 and desaturase gene promote cold tolerance in long-lived Caenorhabditis elegans age-1 mutants. PLoS ONE 6:e245550
Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/ssDNA/PEG procedure. Yeast 11:355–360
Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509
Pereira SL, Leonard AE, Mukerji P (2003) Recent advances in the study of fatty acid desaturases from animals and lower eukaryotes. Prostaglandins Leukot Essent Fatty Acids 68:97–106
Laoteng K, Anjard C, Rachadawong S, Tanticharoen M, Maresca B, Cheevadhanarak S (1999) Mucor rouxii Δ9-desaturase gene is transcriptionally regulated during cell growth and by low temperature. Mol Cell Biol Res Commun 1:36–43
Fukuchi-Mizutani M, Tasaka Y, Tnanka Y, Ashikari T, Kusumi T, Murata N (1998) Characterization of delta 9 acyl-lipid desaturase homologues from Arabidopsis thaliana. Plant Cell Physiol 39:247–253
Wan X, Zhang YB, Wang P, Huang FH, Chen H, Jiang ML (2009) Production of gamma-linolenic acid in Pichia pastoris by expression of a delta-6 desaturase gene from Cunninghamella echinulata. J Microbiol Biotechnol 19:1098–1102
Acknowledgments
This work was supported by National Natural Science Foundation of China (Grants 31000778) and National Hi-Tech Program of China Grant (2011AA100904).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wan, X., Liang, Z., Gong, Y. et al. Characterization of three Δ9-fatty acid desaturases with distinct substrate specificity from an oleaginous fungus Cunninghamella echinulata . Mol Biol Rep 40, 4483–4489 (2013). https://doi.org/10.1007/s11033-013-2540-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-013-2540-4