Highly Unsaturated Fatty Acid Synthesis in Vertebrates: New Insights with the Cloning and Characterisation of a ∆6 Desaturase of Atlantic Salmon

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), 1 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher 2 vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a ∆5 fatty acyl 3 desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally 4 characterized previously. Here we report cloning and functional characterisation of a ∆6 fatty acyl 5 desaturase of Atlantic salmon, and describe its genomic structure, tissue expression and nutritional 6 regulation. A salmon genomic library was screened with a salmon ∆5 desaturase cDNA and 7 positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty 8 acyl desaturase was shown to comprise 2106bp containing an ORF of 1365 bp specifying a protein 9 of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three 10 histidine boxes, two transmembrane regions, and an N-terminal cytochrome b 5 domain containing 11 the haem-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. 12 Functional expression showed that this gene possessed predominantly ∆6 desaturase activity. 13 Screening and sequence analysis of the genomic DNA of a single fish revealed that the ∆6 14 desaturase gene comprised 13 exons in 7965 bp of genomic DNA. Quantitative real time PCR assay 15 of gene expression in Atlantic salmon showed that both ∆6 and ∆5 fatty acyl desaturase genes, and 16 a fatty acyl elongase gene, were highly expressed in intestine, liver and brain, and less so in kidney, 17 heart, gill, adipose tissue, muscle and spleen. Furthermore, expression of both ∆6 and ∆5 fatty acyl 18 desaturase genes in intestine, liver, red muscle and adipose tissue was higher in salmon fed a diet 19 containing vegetable oil than in fish fed a diet containing fish oil. 20 21 22 23 4 Highly unsaturated fatty acids (HUFA), arachidonate (AA; 20:4n-6), eicosapentaenoate (EPA; 23 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), are crucial to the health and normal development 24 of higher vertebrates (1-3). Fish are the most important source of n-3 HUFA for humans, but, with 25 fisheries in decline, an increasing proportion of fish is being provided by rapidly expanding 26 aquaculture (4). Paradoxically, aquaculture is itself dependent upon fisheries for the provision of 27 fishmeals and oils traditionally used in the feed formulations (5). Their use ensured the high 28 nutritional quality of …