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A third fatty acid 9-desaturase from Mortierella alpina with a different substrate specificity to ole1p and ole2p

Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK¹
Roche Products Ltd, Delves Road, Heanor Gate, Heanor, Derbyshire DE75 7SG, UK²
School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK³

Author for correspondence: Donald A. MacKenzie. Tel: +44 1603 255255. Fax: +44 1603 507723. e-mail: donald.mackenzie{at}bbsrc.ac.uk

A third gene (9-3) encoding a fatty acid 9-desaturase was isolated from the oil-producing fungus Mortierella alpina. The predicted protein of 512 aa shared 53% sequence identity with the two fatty acid 9-desaturases, ole1p and ole2p, already described in this organism and contained three histidine boxes, four putative transmembrane domains and a C-terminal cytochrome b₅ fusion that are typical of most fungal membrane-bound fatty acid desaturases. However, unlike the M. alpina ole1 and ole2 genes, the 9-3 ORF failed to complement the Saccharomyces cerevisiae ole1 mutation. GC-MS analysis of fatty-acid-supplemented ole1 yeast transformants containing the 9-3 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 M. alpina fatty acid 9-desaturase genes or the S. cerevisiae OLE1 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 9-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.

Keywords: stearoyl-CoA desaturase, hexacosenoic acid, fatty acid n-9 desaturation, fatty acid elongation, yeast complementation

Abbreviations: 16:0, palmitic acid; 16:1, palmitoleic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, -linolenic acid; -18:3, -linolenic acid; 20:0, arachidic acid; 20:1, eicosenoic acid; 20:3, dihomo--linolenic acid; 20:4, arachidonic acid; 22:0, behenic acid; 22:1, erucic acid; 24:0, lignoceric acid; 24:1, nervonic acid; 26:0, hexacosanoic acid; 26:1, hexacosenoic acid; 9-3, gene encoding fatty acid 9-desaturase homologue; DMOX, 4,4-dimethyloxazoline; ELO2 and ELO3, genes encoding fatty acid elongases; ER, endoplasmic reticulum; FAME, fatty acid methyl ester; LCPUFA, long-chain polyunsaturated fatty acid; ole1 and ole2, genes encoding fatty acid 9-desaturases ole1p and ole2p, respectively; RACE, rapid amplification of cDNA ends; UTR, untranslated region of mRNA

The EMBL accession number for the sequence reported in this paper is AJ278339.

^a Present address: Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Rangsit Center, Patumthanee 12121, Thailand.

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