[Cloning and heterologous expression of a novel delta6 -desaturase gene from Rhizopus arrhizus NK030037].
A 593 bp DNA fragment was amplified from Rhizopus arrhizus NK030037 with degenerate oligonucleotide primers designed based on the sequences information for fungi delta6-fatty acid desaturase genes by RT-PCR and sequenced. Gene specific primers derived from this partial sequence were used for the amplification of the 3'- and 5'-ends of this cDNA by RACE method, and this lead to a full-length cDNA sequence of 1 482 bp was amplified. Sequence analysis showed this cDNA sequence had an open reading frame(ORF) of 1 377 bp coding 458 amino acids of 52 kD. The deduced amino-acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. The full-length cDNA sequence is a putative novel delta6-fatty acid desaturase gene. To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence. The amplified cDNA RAD6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method. Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L. Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees. Total fatty acids were extracted from the induced cells and subjected to methyl-esterification. The resultant fatty acid methyl esters(FAME) were analyzed by gas chromatography(GC). A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new fatty acid to total fatty acids was 3.85%. Gas chromatography-mass spectrometry( GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester. These results showed that the transgenic product exhibited delta6-fatty acid desaurase activity, converting LA to GLA specifically.
Zhang
Qi
Q
Department of Microbiology, Nanakai University, Tianjin 300071, China.
Li
Ming-Chun
MC
Sun
Ying
Y
Ma
Hai-Ting
HT
Ren
Yong
Y
Xing
Lai-Jun
LJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics*; D050602:Linoleoyl-CoA Desaturase; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012233:Rhizopus / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D018076:DNA, Complementary; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase