[Cloning and expression of delta6-desaturase gene from Thamnidium elegans in Saccharomyces cerevisiae].
A 459 bp DNA fragment was amplified from Thamnidium elegans As3.2806 with degenerate oligonucleotides primers designed based on the sequences information from the conserved histidine-rich motifs II and near III of fungal delta6-fatty acid desaturases genes by RT-PCR and sequenced. Gene specific primers designed according to this partial sequence were used for the amplification of the 3'- and 5'- end of this cDNA by RACE method, and sequence information coming from these two ends were used to design two GSPs to clone the 1504bp full-length cDNA. Sequence analysis showed this cDNA sequence had an open reading frame (ORF) of 1377bp encoding 458 amino acids of 52.4kD. The deduced amino acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprise 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. 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 TED6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYTED6, 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 C. Total fatty acids were extracted from the induced cell 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 7.5%. 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 coding product of this sequence exhibited the activity of converting linoleic acid (LA) to gamma-linolenic (GLA), and indicated that amino-acid sequence exhibited delta6-fatty acid desaturase activity.
Wang
De-pei
DP
Tianjin Key Laboratory of Microbial Funitional Genomics, Department of Microbiology, NanKai University, Tianjin 300071, China.
Li
Ming-chun
MC
Wei
Dong-sheng
DS
Zhang
Ying-hui
YH
Xing
Lai-jun
LJ
D000595:Amino Acid Sequence; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008042:Linolenic Acids / Q000032:analysis; D050602:Linoleoyl-CoA Desaturase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009090:Mucorales / Q000201:enzymology / Q000235:genetics*; D016366:Open Reading Frames; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D017931:DNA Primers; D005656:Fungal Proteins; D008042:Linolenic Acids; C047376:methyl linolenate; D050602:Linoleoyl-CoA Desaturase