Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal Δ12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids

We isolated a putative desaturase gene from a marine alga, Pinguiochrysis pyriformis MBIC 10872, which is capable of accumulating eicosapentaenoic acid (C20:5 ^{Δ5,8,11,14,17}). The gene possessed an open reading frame of 1,314 bp encoding a putative 437 amino acid residues showing high sequence identity (37-48) with fungal and nematode Δ12-fatty acid desaturases. Yeast cells transformed with the gene converted endogenous oleic acid (C18:1 ^Δ9) to linoleic acid (C18:2 ^Δ9,12). However, no double bonds were introduced into other endogenous fatty acids or exogenously added fatty acids. Flag-tagged enzyme was recovered in the micosome fraction when expressed in yeast cells. To express the gene in thraustochytrids, a construct driven by the thraustochytrid-derived ubiquitin promoter was used. Interestingly, exogenously added oleic acid was converted to linoleic acid in the gene transformants but not mock transformants of Aurantiochytrium limacinum mh0186. These results clearly indicate that the gene encodes a microsomal Δ12-fatty acid desaturase and was expressed functionally in not only yeasts but also thraustochytrids. This is the first report describing the heterozygous expression of a fatty acid desaturase in thraustochytrids, and could facilitate a genetic approach towards fatty acid synthesis in thraustochytrids which are expected to be an alternative source of polyunsaturated fatty acids.