Production of Gamma-Linolenic Acid in Pichia pastoris by Expression of a Delta-6 Desaturase Gene from Cunninghamella echinulata

摘要

Gamma-linolenic acid (GLA, C18:3 Δ~(6,9,12))is synthesizedby a delta-6 fatty acid desaturase using linoleic acid (LA,C18:2 Δ~(9,1,2))as a substrate. To enable the production ofGLA in the conventional yeast Pichia pastoris, we haveisolated a cDNA encoding the delta-6 fatty acid desaturasefrom Cunninghamella echinulata MIAN6 and confirmedits function by heterogeneous expression in P. pastoris.Sequence analysis indicated that this cDNA sequence hasan open reading frame of 1,404 bp, which encodes a52 kDa peptide of 468 amino acids. This sequence has64% identity to the previously reported delta-6 fatty aciddesaturase from Rhizopus oryzae. The polypeptide has acytochrome b5 domain at the N-terminus including theHPGG motif in the heme-binding region, as reported forother delta-6 fatty acid desaturases. In addition, thisenzyme differs from other desaturases by the presence ofthree possible N-linked glycosylation sites. Analysis of thefatty acid composition demonstrated the accumulationof GLA to the level of 3.1% of the total fatty acids.Notably, the amounts of ginkgolic acid (C17:1) and palmiticacid (C16:0) were increased from 1.3% to 29.6% andfrom 15% to 33%, respectively. These results reveal thatthe modification of the fatty acid biosynthetic pathwayby genetic manipulation in order to produce specificpolyunsaturated fatty acids in P. pastoris is a promisingtechnique.