Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexaenoic acids from alpha-linolenic acid in Atlantic salmon (Salmo salar).
Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b(5) domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an omega-3 delta5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from alpha-linolenic acid (18:3n-3). The desaturase showed only low levels of delta6 activity toward C(18) polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C(18) > C(20) > C(22). Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.
Hastings
Nicola
N
Institute of Aquaculture, University of Stirling, FK9 4LA, Scotland, UK.
Agaba
Morris K
MK
Tocher
Douglas R
DR
Zheng
Xiaozhong
X
Dickson
Cathryn A
CA
Dick
James R
JR
Teale
Alan J
AJ
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004281:Docosahexaenoic Acids / Q000378:metabolism*; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis*; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D000081031:Fatty Acid Elongases; D040461:Gene Components; D015870:Gene Expression; D008969:Molecular Sequence Data; D010802:Phylogeny; D012441:Saccharomyces cerevisiae; D020317:Salmo salar / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017962:alpha-Linolenic Acid / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D017962:alpha-Linolenic Acid; D004281:Docosahexaenoic Acids; D015118:Eicosapentaenoic Acid; D044943:Fatty Acid Desaturases; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases