[Isolation and functional identification of delta 5 desaturase gene from Mortierella alpina]

Arachidonic acid is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes. delta5 desaturase is a key enzyme in the biosynthetic pathway of arachidonic acid, which catalyze the delta5 dehydrogenation of di-homo-gamma-linolenic acid to form arachidonic acid. Complete cDNA encoding putative delta5 fatty acyl desaturase was isolated from Mortierella alpina M6 via RT-PCR. The full length cDNA consisted of 1366 nucleotides encoding 446 amino acids. The deduced protein had conserved domains of known delta5 fatty acyl desaturases including a cytochrome b5 domain in the N terminal and 3 conserved histidine box. To elucidate the function of the protein, the cDNA was subcloned into the expression vector pPIC9K. The resultant recombinant plasmid pPIC9K-D5 was transformed to Pichia pastoris GS115 by electroporation. Transformants containing multi-copy delta5 desaturase gene were screened by Geneticin resistance. The transformants were induced to express the inserted gene with methanol when di-homo-gamma-linolenic acid was provided as an exogenous substrate. Analysis of the recombinant yeast lipids by gas chromatograph showed that a novel peak corresponding to the standard of arachidonic acid was detected. The novel peak was further characterized by GC-MS and identified as arachidonic acid. The results showed that the gene isolated from fungi Mortierella alpina M6 was a delta5 desaturase gene.