Identification and Functional Analysis of a Delta-6 Desaturase from the Marine Microalga Glossomastix chrysoplasta
2007
Hsiao, Tracy Y | Holmes, Bradley | Blanch, Harvey W
A delta-6 (Δ6) desaturase gene was isolated from the marine microalga Glossomastix chrysoplasta, a stramenopile that produces large amounts of eicosapentaenoic acid (EPA). A functional analysis of this gene was performed in Saccharomyces cerevisiae. Isolation of the Δ6 fatty acid desaturase was achieved via reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate primers designed from conserved histidine motifs and 5' and 3' RACE. Two almost identical copies of Δ6 desaturase were found, differing by nine silent mutations. The existence of two such genes may be a result of a recent gene duplication event, or may have arisen from the possible diploid nature of vegetative algae. This appears to be the first instance of two Δ6 desaturase mRNA sequences existing in the same organism. The isolated mRNA sequences and their corresponding hypothetical protein, GcDES6, were found to contain features characteristic of a membrane-bound Δ6 desaturase, including membrane-spanning regions separating conserved histidine boxes and N-terminal cytochrome b5 fusion. Heterologous expression in S. cerevisiae was used to confirm Δ6 regioselectivity and the function of GcDES6. Both ω3(18:3Δ9,12,15) and ω6(18:2Δ9,12) precursors can be used by GcDES6 in vivo with similar desaturase activity. One intron site was found in the cytochrome b5 fusion region of GcDES6. Although the conservation of intron-exon junctions has been found for several desaturases in humans and in Caenorhabditis elegans, a comparison of introns in GcDES6 and other Δ6 desaturases has not revealed any strong similarities.
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