Production of gamma-linolenic acid in Pichia pastoris by Expression of a delta-6 desaturase gene from Cunninghamella echinulata.
Gamma-linolenic acid (GLA, C18:3 delta(6,9,12)) is synthesized by a delta-6 fatty acid desaturase using linoleic acid (LA, C18:2 delta(9,12)) as a substrate. To enable the production of GLA in the conventional yeast Pichia pastoris, we have isolated a cDNA encoding the delta-6 fatty acid desaturase from Cunninghamella echinulata MIAN6 and confirmed its function by heterogeneous expression in P. pastoris. Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,404 bp, which encodes a 52 kDa peptide of 468 amino acids. This sequence has 64% identity to the previously reported delta-6 fatty acid desaturase from Rhizopus oryzae. The polypeptide has a cytochrome b5 domain at the N-terminus including the HPGG motif in the heme-binding region, as reported for other delta-6 fatty acid desaturases. In addition, this enzyme differs from other desaturases by the presence of three possible N-linked glycosylation sites. Analysis of the fatty acid composition demonstrated the accumulation of GLA to the level of 3.1% of the total fatty acids. Notably, the amounts of ginkgolic acid (C17:1) and palmitic acid (C16:0) were increased from 1.3% to 29.6% and from 15% to 33%, respectively. These results reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in P. pastoris is a promising technique.
Wan
Xia
X
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, 430062 Wuhan, P. R.China.
Zhang
Yinbo
Y
Wang
Ping
P
Huang
Fenghong
F
Chen
Hong
H
Jiang
Mulan
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D020093:Cunninghamella / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017965:gamma-Linolenic Acid / Q000378:metabolism*
D005656:Fungal Proteins; D017965:gamma-Linolenic Acid; D050602:Linoleoyl-CoA Desaturase