Xiaozhong Zheng

Highly unsaturated fatty acid synthesis in vertebrates : New insights with the cloning and characterization of a Δ6 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 Δ5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterization of a Δ6 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 Δ5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame 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 heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly Δ6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the Δ6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both Δ6 and Δ5 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 Δ6 and Δ5 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.

Molecular Cloning and Functional Characterization of Fatty Acyl Desaturase and Elongase cDNAs Involved in the Production of Eicosapentaenoic and Docosahexaenoic Acids from α -Linolenic Acid in Atlantic Salmon ( Salmo salar )

Fish are the only major dietary source for humans of ω-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 b5 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 ω-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 toward C18 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 C18 > C20 > C22. 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.

Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional regulation of fatty acyl Δ6 desaturase of Atlantic cod (Gadus morhua L.)

This study reports the cloning, functional characterization, tissue expression, and nutritional regulation of a Δ6 fatty acyl desaturase of Atlantic cod (Gadus morhua). PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from cod liver, with full-length cDNA obtained by rapid amplification of cDNA ends. The cDNA for the putative desaturase was shown to comprise 1980 bp, including a 261-bp 5′-UTR, a 375-bp 3′-UTR, and 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 heme-binding motif HPGG, all characteristic of microsomal fatty acyl desaturases. The cDNA displayed Δ6 desaturase activity in a yeast expression system. Quantitative real-time PCR assay of gene expression in cod showed that the Δ6 desaturase gene was expressed highly in brain, to a slightly lesser extent in liver, kidney, intestine, red muscle, and gill, and at much lower levels in white muscle, spleen, and heart. The expression of the Δ6 desaturase gene did not appear to be under significant nutritional regulation, with levels in liver and intestine being barely altered in fish fed a vegetable oil blend, in comparison with levels in fish fed fish oil. This was reflected in enzyme activity, as hepatocytes or enterocytes showed very little highly unsaturated FA biosynthesis activity irrespective of diet. Further studies are required to determine why the Δ6 desaturase appears to be barely functional in cod under the conditions tested.

Highly Unsaturated Fatty Acid Synthesis in Atlantic Salmon: Characterization of ELOVL5- and ELOVL2-like Elongases

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 complementary DNA (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-amino-acid (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 zebra fish, Acanthopterygian fish species lack elovl2 which is consistent with their negligible ability to biosynthesize 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 biosynthesize LC-PUFA and may provide a biotechnological tool to produce enhanced levels of LC-PUFA, particularly DHA, in transgenic organisms.

Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and Delta6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish physiology.

Long-chain polyunsaturated fatty acid synthesis in fish: Comparative analysis of Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.) Δ6 fatty acyl desaturase gene promoters

Fish vary in the ability to biosynthesise n-3 long-chain polyunsaturated fatty acids (LC-PUFA), with marine fish such as cod being inefficient in comparison to freshwater and salmonid fish. We investigated differences in the gene promoters of Delta6 fatty acyl desaturase (Delta6 FAD), a critical enzyme in LC-PUFA biosynthesis, in cod and salmon. Progressive deletions and targeted mutations of the promoters were tested for activity in a transfected Atlantic salmon (AS) cell line under low or high LC-PUFA treatment, and regions sufficient to direct transcription were identified. Comparison of these regions with sequences of corresponding regions of Delta6 FAD genes from mammals, amphibians and fish indicated a remarkable conservation of binding sites for SREBPs and NF-Y. In addition to these sites, a site was identified in salmon with similarity to that recognised by Sp1 transcription factor, and which was required for full expression of the salmon Delta6 FAD gene. The cod promoter was less active and lacked the Sp1 site. Eicosapentaenoic acid suppressed LC-PUFA synthesis in AS cells and also suppressed activity of the salmon Delta6 FAD promoter although this activity was likely mediated through sites other than Sp1, possibly similar to those recognised by NF-Y and SREBP transcription factors.

Influence of dietary oil content and conjugated linoleic acid (CLA) on lipid metabolism enzyme activities and gene expression in tissues of atlantic salmon (Salmo salar L.)

The overall objective is to test the hypothesis that conjugated linoleic acid (CLA) has beneficial effects in Atlantic salmon as a result of affecting lipid and FA metabolism. The specific aims of the present study were to determine the effects of CLA on some key pathways of FA metabolism, including FA oxidation and highly unsaturated FA (HUFA) synthesis. Salmon smolts were fed diets containing two levels of fish oil (low, ∼17%, and high, ∼34%) containing three levels of CLA (a 1∶1 mixture of cis-9,trans-11 and trans-10,cis-12 at 0, 1, and 2% of diet) for 3 mon. The effects of dietary CLA on HUFA synthesis and β-oxidation were measured, and the expression of key genes in the FA oxidation and HUFA synthesis pathways, and the potentially important transcription factors peroxisome proliferators activated receptors (PPAR), were determined in selected tissues. Liver HUFA synthesis and desaturase gene expression was increased by dietary CLA and decreased by high diatary oil content. Carnitine palmitoyltransferase-I (CPT-I) activity and gene expression were generally increased by CLA in muscle tissues although they were relatively unaffected by dietary oil content. In general CPT-1 activity or gene expression was not correlated with β-oxidation. Dietary CLA tended to increase PPARα and β gene expression in both liver and muscle tissues, and PPARγ in liver. In summary, gene expression and activity of the FA pathways were altered in response to dietary CLA and/or oil content, with data suggesting that PPAR are also regulated in response to CLA. Correlations were observed between dietary CLA, liver HUFA synthesis and desaturase gene expression, and liver PPARα expression, and also between dietary CLA, CPT-I expression and activity, and PPARα expression in muscle tissues. In conclusion, this study suggests that dietary CLA has effects on FA metabolism in Atlantic salmon and on PPAR transcription factors. However, further work is required to assess the potential of CLA as a dietary supplement, and the role of PPAR in the regulation of lipid metabolism in fish.

Regulatory divergence of homeologous Atlantic salmon elovl5 genes following the salmonid-specific whole genome duplication

Fatty acyl elongase 5 (elovl5) is a critical enzyme in the vertebrate biosynthetic pathway which produces the physiologically essential long-chain polyunsaturated fatty acids (LC-PUFA), docosahexenoic acid (DHA), and eicosapentenoic acid (EPA) from 18 carbon fatty acids precursors. In contrast to most other vertebrates, Atlantic salmon possess two copies of elovl5 (elovl5a and elovl5b) as a result of a whole-genome duplication (WGD) which occurred at the base of the salmonid lineage. WGDs have had a major influence on vertebrate evolution, providing extra genetic material, enabling neofunctionalization to accelerate adaptation and speciation. However, little is known about the mechanisms by which such duplicated homeologous genes diverge. Here we show that homeologous Atlantic salmon elovl5a and elovl5b genes have been asymmetrically colonised by transposon-like elements. Identical locations and identities of insertions are also present in the rainbow trout duplicate elovl5 genes, but not in the nearest extant representative preduplicated teleost, the northern pike. Both elovl5 salmon duplicates possessed conserved regulatory elements that promoted Srebp1- and Srebp2-dependent transcription, and differences in the magnitude of Srebp response between promoters could be attributed to a tandem duplication of SRE and NF-Y cofactor binding sites in elovl5b. Furthermore, an insertion in the promoter region of elovl5a confers responsiveness to Lxr/Rxr transcriptional activation. Our results indicate that most, but not all, transposon mobilisation into elovl5 genes occurred after the split from the common ancestor of pike and salmon, but before more recent salmonid speciations, and that divergence of elovl5 regulatory regions have enabled neofuntionalization by promoting differential expression of these homeologous genes.