Running title: Characterisation of salmon ELOVL5-and ELOVL2 elongases Abbreviations: aa, amino acid; ARA, arachidonic acid (20:4n-6); DHA, docosahexaenoic acid (22:6n-3); EPA, eicosapentaenoic acid (20:5n-3); ER, endoplasmic reticulum; FA, fatty acid; FO, fish oil; HUFA, highly unsaturated fatty acids (carbon chain length ≥ C 20 with ≥ 3 double bonds); LO, linseed oil; ORF, open reading frame; PUFA, polyunsaturated fatty acids; Q-PCR, quantitative (real-time) polymerase chain reaction; RACE, rapid amplification of cDNA ends; RO, rapeseed oil; SO, soybean oil; UTR, untranslated region; VO, vegetable oil. 2Abstract Fish species vary in their capacity to biosynthesize the n-3 long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of LC-PUFA involves enzyme-mediated fatty acyl desaturation and elongation. Previously, a cDNA for an elongase, now termed elovl5a, had been cloned from Atlantic salmon. Here we report on the cloning of two new elongase cDNAs: a second elovl5b elongase, corresponding to a 294 aa protein, and an elovl2-like elongase, coding for a 287 aa protein, characterized for the first time in a nonmammalian vertebrate. Heterologous expression in yeast showed that the salmon Elovl5b elongated C18 and C20 PUFA, with low activity towards C22, while Elovl2 elongated C20 and C22 PUFA with lower activity towards C18 PUFA. All three transcripts showed predominant expression in the intestine and liver, followed by the brain. Elongase expression showed differential nutritional regulation. Levels of elovl5b and particularly of elovl2, but not of elovl5a, transcripts were significantly increased in liver of salmon fed vegetable oils (VO) compared to fish fed fish oil (FO). Intestinal expression showed a similar pattern.Phylogenetic comparisons indicate that, in contrast to salmon and zebrafish, Acanthopterygian fish species lack elovl2 which is consistent with their neglible ability to biosynthesise LC-PUFA and to adapt to VO dietary inclusion, compared to predominantly freshwater salmonids. Thus the presence of elovl2 in salmon explains the ability of this species to biosynthesise LC-HUFA and may provide a biotechnological tool to produce enhanced levels of LC-PUFA, particularly DHA, in transgenic organisms.3

Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 18∶2n − 6 to produce 18∶3n − 6 that is elongated to 20∶3n − 6 followed by Δ5 desaturation. Synthesis of EPA from 18∶3n − 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6∕Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22∶5n − 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22∶5n − 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above.Δ4 desaturase | bifunctional Δ6/Δ5 desaturase | polyunsaturated fatty acid biosynthesis | Siganus canaliculatus | teleost

Highly unsaturated fatty acid (HUFA) synthesis in Atlantic salmon (Salmo salar) was known to be influenced by both nutritional and environmental factors. Here we aimed to test the hypothesis that both these effectors involved similar molecular mechanisms. Thus, HUFA biosynthetic activity and the expression of fatty acyl desaturase and elongase genes were determined at various points during an entire two year production cycle in salmon fed diets containing either 100% fish oil or diets in which a high proportion (75% and 100%) of fish oil was replaced by C 18 polyunsaturated fatty acid-rich vegetable oil. The results showed that HUFA biosynthesis in Atlantic salmon varied during the growth cycle with peak activity around seawater transfer and subsequent low activities in seawater. Consistent with this, gene expression of Δ6 desaturase, the rate-limiting step in the HUFA biosynthetic pathway, was highest around the point of seawater transfer and lowest during the seawater phase. In addition, the expression of both Δ6 and Δ5 desaturase genes was generally higher in fish fed the vegetable oilsubstituted diets compared to fish fed fish oil, particularly in the seawater phase. Again, generally consistent with this, the activity of the HUFA biosynthetic pathway was invariably higher in fish fed diets in which fish oil was substituted by vegetable oil compared to fish fed only fish oil. In conclusion, these studies showed that both nutritional and environmental modulation of HUFA biosynthesis in Atlantic salmon involved regulation of fatty acid desaturase gene expression.3

Fig. 7The first diglyceride produced by PtdCho should have contained an oxygen atom, thus the vertical dashed line from the CH 2 -O bond has been moved to the O-P bond. The same is true for PtdOH, so the vertical dashed line from the P-O bond has been moved to the O-CH 2 bond. For Lyso-PtdCho (16:0), the vertical dashed line from the O-CH bond has been moved to the O=C-O bond. In addition, the carbon number of EPA has been corrected to 20, instead of 22, near the CH 2 -C=O bond.

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFA) 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. A greater understanding of how fish metabolise and biosynthesise HUFA may lead to effective use of 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 optimising the use of vegetable oils in Atlantic salmon culture. In this paper we 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, all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3 are displayed by this one polypeptide product.