Isolation and characterization of a Δ5 FA desaturase from Pythium irregulare by heterologous expression in Saccharomyces cerevisiae and oilseed crops

Abstract

By using the polymerase chain reaction approach with two degenerate primers targeting the heme-binding and the third histidine-rich motifs in microsomal carboxyl-directed desaturases, we identified a cDNA PiD5 from Pythium irregulare encoding a Δ5 desaturase. The substrate specificity of the enzyme was studied in detail by expressing PiD5 in a yeast (Saccharomyces cerevisiae) mutant strain, AMY-2α, where ole1, a Δ9 desaturase gene, is disrupted. The result revealed that the encoded enzyme could desaturate unsaturated FA from 16 to 20 carbons beginning with Δ9 and Δ11 as well as Δ8 ethylenic double bonds. Introduction of PiD5 into Brassica juncea under the control of a CaMV 35S constitutive promoter resulted in accumulation of several Δ5-unsaturated polymethylene-interrupted FA (Δ5-UPIFA) including 18:2-5,9, 18:2-5,11, 18:3-5,9,12, and 18:4-5,9,12,15 in vegetative tissues. The transgenic enzyme could also desaturate the exogenously supplied homo-γ-linolenic acid (20:3-8,11,14) to arachidonic acid (20:4-5,8,11,14). Introduction of PiD5 into B. juncea and flax under the control of seed-specific promoters resulted in production of Δ5-UPIFA, representing more than 10% of the total FA in the seeds.