A novel fungal ?3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4

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Abstract
A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4n-6) but also eicosapentaenoic acid (EPA; 20:5n-3) below a cultural temperature of 20°C. Here, we describe the isolation and characterization of a gene (maw3) that encodes a novel ω3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 Δ12-desaturase and Saccharomyces kluyveri ω3-desaturase, the ω3-desaturase gene from M. alpina 1S-4 was cloned. Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M. alpina 1S-4 Δ12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri ω3-desaturase. The cloned cDNA was confirmed to encode the ω3-desaturase by its expression in the yeast Sac. cerevisiae. Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs. The substrate specificity of the M. alpina 1S-4 ω3-desaturase differs from those of the known fungal ω3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri ω3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina ω3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans ω3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 ω3-desaturase is rather similar to that of C. elegans ω3-desaturase, but the M. alpina ω3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 ω3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.

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