Identification and functional analysis of a delta-6 desaturase from the marine microalga Glossomastix chrysoplasta.
A delta-6 (Delta6) 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 Delta6 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 Delta6 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 Delta6 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 Delta6 desaturase, including membrane-spanning regions separating conserved histidine boxes and N-terminal cytochrome b5 fusion. Heterologous expression in S. cerevisiae was used to confirm Delta6 regioselectivity and the function of GcDES6. Both omega3(18:3Delta9,12,15) and omega6(18:2Delta9,12) 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 Delta6 desaturases has not revealed any strong similarities.
Hsiao
Tracy Y
TY
Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA.
Holmes
Bradley
B
Blanch
Harvey W
HW
D000595:Amino Acid Sequence; D017931:DNA Primers / Q000737:chemistry; D056890:Eukaryota / Q000201:enzymology* / Q000235:genetics*; D005227:Fatty Acids / Q000032:analysis / Q000096:biosynthesis; D050602:Linoleoyl-CoA Desaturase / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D022821:RNA Splice Sites / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment
D017931:DNA Primers; D005227:Fatty Acids; D022821:RNA Splice Sites; D050602:Linoleoyl-CoA Desaturase