Cloning of Δ8-fatty acid desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae

Abstract:

Δ8 desaturase pathway, different from common Δ6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Δ8-fatty acid desaturase is one of the key enzymes in Δ8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn’t intron in this Δ8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning Δ8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates ω6-eicosadienoic acid and ω3-eicosatrienoic acid for the expression of Δ8-fatty acid desaturase gene. The results indicated that high level expressed Δ8-fatty acid desaturase could convert ω6-eicosadienoic acid and ω3-eicosatrienoic acid to dihomo-γ-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.