Cloning and expression of a △6-fatty acid desaturase gene from Rhizopus stolonifer in Saccharomyces cervisiae

Abstract:

Fatty acid composition of fungi is analysed through the gas chromatography(GC) technique. With specific activity a novel enzyme △6-fatty acid desaturase was screened and isolated from Rhizopus stolonifer. In this study R.stolonifer was identified as a fungal species that produced plentiful γ-linolenic acid. A 1475bp full-length cDNA, designated as RnD6Dhere, with high homology to fungal △6-fatty acid desaturase genes was isolated from R. stolonifer 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 1380bp 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. To elucidate the function of his novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to γ-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 GLA was 12.25% of total fatty acids. The result demonstrated that the coding produced △6-fatty acid desaturase activity of RnD6D which led to the accumulation of γ-linolenic acid.