Identification and characterization of a novel delta6-fatty acid desaturase gene from Rhizopus nigricans.
Fatty acid composition of fungi is analysed through the gas chromatography technique. With specific activity a novel enzyme Delta6-fatty acid desaturase was screened and isolated from Rhizopus nigricans. In this study R. nigricans was identified as a fungal species that produced plentiful gamma-linolenic acid. A 1,475 bp full-length cDNA, designated as RnD6D here, with high homology to fungal Delta6-fatty acid desaturase genes was isolated from R. nigricans using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1,380 bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. The corresponding genomic sequence of RnD6D was 1,689 bp with 4 introns, which was at least one intron more than other fungal Delta6-fatty acid desaturase genes. To elucidate the function of this novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to gamma-linolenic acid methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The result demonstrated that the coding produced Delta6-fatty acid desaturase activity of RnD6D which led to the accumulation of gamma-linolenic acid. The functionally active RnD6D gene cloned here defined a novel Delta6-fatty acid desaturase from R. nigricans.
Lu
He
H
Department of Microbiology, Chong Qing Medica University, 400016 Chongqing, People's Republic of China. lhluhe@163.com
Li
Jia-Na
JN
Chai
You-Rong
YR
Zhang
Xue-Kun
XK
D000595:Amino Acid Sequence; D001483:Base Sequence; D008401:Gas Chromatography-Mass Spectrometry; D005816:Genetic Complementation Test; D057927:Hydrophobic and Hydrophilic Interactions; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017965:gamma-Linolenic Acid / Q000378:metabolism*
D017965:gamma-Linolenic Acid; D050602:Linoleoyl-CoA Desaturase