Biochemical and molecular studies of the polyunsaturated fatty acid desaturation pathway in fish

Fish have an absolute dietary requirement for certain polyunsaturated fatty acids (PUFA) termed “essential fatty acids” (EFA) that include members of both the n-6 and n-3 series typified by linoleic acid, 18:2n-6, and α-linolenic acid, 18:3n-3. However, the biologically active forms of EFA are generally the C20 and C22 metabolites of 18:2n-6 and 18:3n-3, viz. 20:4n-6, 20:5n-3 and 22:6n-3. Some fish species can convert C18 PUFA to the C20 and C22 PUFA through a series of alternating desaturation and chain elongation reactions mediated by microsomal systems containing elongases and Δ6 and Δ5 fatty acid desaturases. In species that cannot perform these conversions, the C20 and C22 PUFA themselves are dietary EFA and their C18 homologues do not satisfy EFA requirements. The extent to which the foregoing statements apply quantitatively to a given fish species varies widely. Therefore, a vital area in lipid nutrition in fish is the provision of sufficient amounts of the correct EFA to satisfy the requirements for normal growth and development, requirements that can vary quantitatively during the life of the fish and are particularly important factors in larval marine fish. This paper reviews the work on defining and characterising the fatty acid desaturation and elongation pathway in fish. Biochemical studies have been advanced by the use of cell cultures which have elucidated key parts of the pathway. Thus, the presence of the so-called Sprecher shunt, where 22:6n-3 is produced from 20:5n-3 through two successive elongations and a Δ6 desaturase followed by peroxisomal chain shortening, was demonstrated in trout. Similarly, the block in the pathway in marine and/or piscivorous fish could be due to either a deficiency of C18-20 elongase or Δ5 desaturase and this varies between different marine species. Recent work has focussed on the molecular biology of the pathway with the cloning of fatty acid desaturases and elongases from a variety of fish species. Zebrafish have been used as a model species and a unique desaturase possessing both Δ6 and Δ5 activity along with an elongase with very high C18-20 activity have been cloned and characterised. Understanding this pathway is of increased importance due to the current dependence of salmonid and marine fish aquaculture on fish oil, the supply of which is becoming increasingly limited and unsustainable, necessitating the use in fish feeds of sustainable plant oils, rich in C18 PUFA, but devoid of C20 and C22 PUFA

Effects of diets containing vegetable oil on expression of genes involved in highly unsaturated fatty acid biosynthesis in liver of Atlantic salmon (Salmo salar)

Fish are an important dietary source of the long-chain C20 and C22 highly unsaturated fatty acids (HUFA), arachidonate (20:4n-6), eicosapentaenoate (20:5n-3) and docosahexaenoate (22:6n-3), that are crucial to the health of higher vertebrates and that can be beneficial in human diets. Δ5 and Δ6 fatty acid desaturases, and fatty acid elongases are critical enzymes in the biosynthetic pathways of HUFA from shorter chain C18 polyunsaturated fatty acids (PUFA) such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids. Recently, full-length cDNAs for fatty acid desaturase and elongase enzymes have been cloned from Atlantic salmon. Functional characterisation of the desaturase revealed n-3 Δ5 desaturase activity, whereas the elongase had broad substrate specificity for PUFA with a range of chain lengths from C18 to C22. The study described here was primarily focused on the nutritional regulation of genes involved in the HUFA biosynthetic pathway in Atlantic salmon. A feeding trial was performed whereby salmon smolts in seawater pens were fed for 40 weeks on five different diets. The diets consisted of a control diet containing fish oil (FO) and four diets in which the FO was replaced in a graded manner by linseed oil (LO). Specifically, in terms of added oils, the five diets were 100% FO (FO), 100%LO (LO100) and FO/LO in ratios of 3:1 (LO25), 1:1 (LO50) and 1:3 (LO75). Fish were sampled at 20 and 40 weeks, and samples of liver were collected for lipid analyses and total RNA extraction. Hepatocytes were also prepared and the activity of the HUFA biosynthetic pathway determined. Expression of fatty acid desaturase and elongase genes was determined by quantitative real time PCR and the ratio of the copy number of the targeted gene against that of β-actin was calculated. The results showed that after 20 weeks of feeding, desaturase and elongase gene expression in liver was increased in a graded manner by increasing dietary LO. Expression of both genes was positively and negatively correlated with dietary 18:3n-3 and n-3 HUFA, respectively. By 40 weeks of feeding, expression of neither gene showed the same degree of correlation with dietary fatty acid composition. In contrast, activity of the HUFA biosynthetic pathway, which showed some association with diet at 20 weeks, was positively and significantly correlated with dietary LO after 40 weeks of feeding. Elongation activity reflected the overall activity of the HUFA biosynthetic pathway to a greater degree than Δ5 desaturation activity. The possible mechanisms underlying the observed results and the regulation of the HUFA biosynthetic pathway are discussed

Highly unsaturated fatty acid synthesis in vertebrates: new insights with the cloning and characterization of a delta6 desaturase of Atlantic salmon

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a ∆5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterisation of a ∆6 fatty acyl desaturase of Atlantic salmon, and describe its genomic structure, tissue expression and nutritional regulation. A salmon genomic library was screened with a salmon ∆5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106bp containing an ORF of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the haem-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly ∆6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the ∆6 desaturase gene comprised 13 exons in 7965 bp of genomic DNA. Quantitative real time PCR assay of gene expression in Atlantic salmon showed that both ∆6 and ∆5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver and brain, and less so in kidney, heart, gill, adipose tissue, muscle and spleen. Furthermore, expression of both ∆6 and ∆5 fatty acyl desaturase genes in intestine, liver, red muscle and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil

Zebrafish cDNA encoding multifunctional fatty acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids

Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids. The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina. A search of the Genbank database identified several EST sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO. The full-length transcript, ZfELO, encoding a polypeptide of 291 amino acid residues was isolated from zebrafish liver cDNA. The predicted amino acid sequence of the open reading frame (ORF) shared high similarity with the elongases of C. elegans and human. When expressed in Saccharomyces cerevisiae, the zebrafish ORF conferred the ability to lengthen the chain of a range of C18, C20 and C22 polyunsaturated fatty acids, indicating that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is not only feasible, but that one elongase enzyme can perform all three elongation steps required. The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously

Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis niloticus)

The desaturation and elongation of [1-14C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level

Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexanoic 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 (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 describe the cloning and functional characterisation of two genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase cDNA, SalDes, was isolated that included an open reading frame (ORF) of 1362 bp specifying a protein of 454 amino acids. The protein sequence included all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing a haem-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast, Saccharomyces cerevisiae, showed SalDes is predominantly an omega-3 Δ5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from α-linolenic acid (18:3n-3). The desaturase showed only low levels of Δ6 activity towards C18 polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that includes an ORF of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo includes characteristic features 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 C18 > C20 > C22. 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

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr–smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)–salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community

Cloning and functional characterisation of polyunsaturated fatty acid elongases of marine and freshwater teleost fish

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids are key to the biosynthesis of the highly unsaturated fatty acids, arachidonic, eicosapentaenoic and docosahexaenoic acids from linoleic and α-linolenic acids. A Mortierella alpina cDNA polyunsaturated fatty acid elongase sequence identified mammalian, amphibian, zebrafish and insect expressed sequence tags (ESTs) in GenBank. Consensus primers were designed in conserved motifs and used to isolate full length cDNA from livers of several fish species by Rapid Amplification of cDNA Ends (RACE). The amplified cDNAs encoded putative open reading frames (ORFs) of 288-294 amino acids that were highly conserved among the fish species. Heterologous expression in yeast, Saccharomyces cerevisiae, demonstrated that all of the ORFs encoded elongases with the ability to lengthen polyunsaturated fatty acid substrates with chain lengths from C18 to C22 and also monounsaturated fatty acids, but not saturated fatty acids. There were differences in the functional competence of the elongases from different fish species. Most of the fish elongases showed a pattern of activity towards different fatty acid substrates in the rank order C18 > C20 >C22, although the tilapia and turbot elongases had similar activity towards 18:4n-3 and 20:5n-3. The fish elongases generally showed greater activity or similar activities with n-3 than with n-6 homologues, with the exception of the cod enzyme which was more active towards n-6 fatty acids

Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional regulation of fatty acyl delta6 desaturase of Atlantic cod (Gadus morhua L.)

Fish contain high levels of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. Biosynthesis of HUFA requires enzyme-mediated desaturation of fatty acids. Here we report cloning and functional characterisation of a ∆6 fatty acyl desaturase of Atlantic cod (Gadus morhua), and describe its tissue expression and nutritional regulation. PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from liver of cod. The full-length cDNA was obtained by Rapid Amplification of cDNA Ends (RACE). The cDNA for the putative fatty acyl desaturase was shown to comprise 1980bp which included a 5’-UTR of 261bp and a 3’-UTR of 375bp. Sequencing revealed that the cDNA included an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the haem-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. The cDNA displayed Δ6 desaturase activity in a heterologous yeast expression system. Quantitative real time PCR assay of gene expression in cod showed that the ∆6 desaturase gene, was highly expressed in brain, relatively highly expressed in liver, kidney, intestine, red muscle and gill, and expressed at much lower levels in white muscle, spleen and heart. In contrast, the abundance of a cod fatty acyl elongase transcript was high in brain and gill, with intermediate levels in kidney, spleen, intestine and heart, and relatively low expression in liver. The expression of the Δ6 desaturase gene and the PUFA elongase gene may be under a degree of nutritional regulation, with levels being marginally increased in livers and intestine of fish fed a vegetable oil blend by comparison with levels in fish fed fish oil. However, this was not reflected in increased Δ6 desaturase activity in hepatocytes or enterocytes, which showed very little highly unsaturated fatty acid biosynthesis activity irrespective of diet. The study described has demonstrated that Atlantic cod express a fatty acid desaturase gene with functional Δ6 activity in a yeast expression system. This is consistent with an established hypothesis that the poor ability of marine fish to synthesise HUFA is not due to lack of a Δ6 desaturase, but rather to deficiencies in other parts of the biosynthetic pathway. However, further studies are required to determine why the Δ6 desaturase appears to be barely functional in cod under the conditions tested