Óscar Monroig

Vertebrate fatty acyl desaturase with Δ4 activity

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.

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.

Biosynthesis of Polyunsaturated Fatty Acids in Marine Invertebrates: Recent Advances in Molecular Mechanisms

Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs.

Expression of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis genes during zebrafish Danio rerio early embryogenesis

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in important physiological processes, many of which are particularly vital during embryonic development. This study investigated the expression of genes encoding enzymes involved in LC-PUFA biosynthesis, namely fatty acyl desaturase (Fad) and Elovl5- and Elovl2-like elongases, during early embryonic development of zebrafish. First, zebrafish elovl2 cDNA was isolated and functionally characterised in yeast, showing high specificity towards C20- and C22-PUFAs, compared to C18 substrates. Second, spatial-temporal expression for elovl2 and the previously cloned fad and elovl5 were studied during zebrafish early embryonic development. Temporal expression shows that all three genes are expressed from the beginning of embryogenesis (zygote), suggesting maternal mRNA transfer to the embryo. However, a complete activation of the biosynthetic pathway seems to be delayed until 12 hpf, when noticeable increases of fad and elovl2 transcripts were observed, in parallel with high docosahexaenoic acid levels in the embryo. Spatial expression was studied by whole-mount in situ hybridisation in 24 hpf embryos, showing that fad and elovl2 are highly expressed in the head area where neuronal tissues are developing. Interestingly, elovl5 shows specific expression in the pronephric ducts, suggesting an as yet unknown role in fatty acid metabolism during zebrafish early embryonic development. The yolk syncytial layer also expressed all three genes, suggesting an important role in remodelling of yolk fatty acids during zebrafish early embryogenesis. Tissue distribution in zebrafish adults demonstrates that the target genes are expressed in all tissues analysed, with liver, intestine and brain showing the highest expression.

Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass, Lates calcarifer

Lates calcarifer, commonly known as the Asian sea bass or barramundi, is an interesting species that has great aquaculture potential in Asia including Malaysia and also Australia. We have investigated essential fatty acid metabolism in this species, focusing on the endogenous highly unsaturated fatty acid (HUFA) synthesis pathway using both biochemical and molecular biological approaches. Fatty acyl desaturase (Fad) and elongase (Elovl) cDNAs were cloned and functional characterization identified them as ∆6 Fad and Elovl5 elongase enzymes, respectively. The ∆6 Fad was equally active toward 18:3n-3 and 18:2n-6, and Elovl5 exhibited elongation activity for C18–20 and C20–22 elongation and a trace of C22–24 activity. The tissue profile of gene expression for ∆6 fad and elovl5 genes, showed brain to have the highest expression of both genes compared to all other tissues. The results of tissue fatty acid analysis showed that the brain contained more docosahexaenoic acid (DHA, 22:6n-3) than flesh, liver and intestine. The HUFA synthesis activity in isolated hepatocytes and enterocytes using [1-14C]18:3n-3 as substrate was very low with the only desaturated product detected being 18:4n-3. These findings indicate that L. calcarifer display an essential fatty acid pattern similar to other marine fish in that they appear unable to synthesize HUFA from C18 substrates. High expression of ∆6 fad and elovl5 genes in brain may indicate a role for these enzymes in maintaining high DHA levels in neural tissues through conversion of 20:5n-3.

Long-chain polyunsaturated fatty acid biosynthesis in chordates: Insights into the evolution of Fads and Elovl gene repertoire.

Long-chain polyunsaturated fatty acids (LC-PUFA) are major components of complex lipid molecules and are also involved in numerous critical biological processes. Studies conducted mainly in vertebrates have demonstrated that LC-PUFA can be biosynthesized through the concerted action of two sets of enzymes, namely fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl) proteins. While LC-PUFA research is a thriving field, mainly focused on human health, an integrated view regarding the evolution of LC-PUFA biosynthetic genetic machinery in chordates is yet to be produced. Particularly important is to understand whether lineage specific life history trajectories, as well as major biological transitions, or particular genomic processes such as genome duplications have impacted the evolution of LC-PUFA biosynthetic pathways. Here we review the gene repertoire of Fads and Elovl in chordate genomes and the diversity of substrate specificities acquired during evolution. We take advantage of the magnitude of genomic and functional data to show that combination duplication processes and functional plasticity have generated a wide diversity of physiological capacities in extant lineages. A clear evolutionary framework is provided, which will be instrumental for the full clarification of functional capacities between the various vertebrate groups.

Functional desaturase fads1 (δ5) and fads2 (δ6) orthologues evolved before the origin of jawed vertebrates

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales.

Expression and role of Elovl4 elongases in biosynthesis of very long-chain fatty acids during zebrafish Danio rerio early embryonic development

Elovl4 is a fatty acyl elongase that participates in the biosynthesis of very long-chain fatty acids (>/=C24), which are relatively abundant in skin (saturated chains), or retina, brain and testes (polyunsaturated chains) of mammals. In the present study we characterised two Elovl4 proteins, Elovl4a and Elovl4b, from zebrafish Danio rerio, and investigated their expression patterns during embryonic development. Heterologous expression in bakers yeast showed that both zebrafish Elovl4 proteins efficiently elongated saturated fatty acids up to C36, with 26:0 appearing the preferred substrate as reported for human ELOVL4. Interestingly, activity for the elongation of PUFA substrates was only shown by Elovl4b, which effectively converted eicosapentaenoic (20:5n-3) and arachidonic (20:4n-6) acids to elongated polyenoic products up to C36. Furthermore, zebrafish Elovl4b may be involved in the biosynthesis of docosahexaenoic acid (22:6n-3, DHA) as it had the capacity to elongate 22:5n-3 to 24:5n-3 which can be subsequently desaturated and chain shortened to DHA in peroxisomes. The distinct functional roles of zebrafish Elovl4 proteins were also reflected in their spatial-temporal expression patterns during ontogeny. Analyses by whole-mount in situ hybridisation in zebrafish embryos showed that elovl4a was expressed in neuronal tissues (wide-spread distribution in the head area), with elovl4b specifically expressed in epiphysis (pineal gland) and photoreceptor cells in the retina. Similarly, tissue distribution in adults revealed that elovl4a transcripts were found in most tissues analysed, whereas elovl4b expression was essentially restricted to eye and gonads. Overall, the results suggest that zebrafish elovl4b resembles other mammalian orthologues in terms of function and expression patterns, whereas elovl4a may represent an alternative elongase not previously described in vertebrates.

Identification of a Δ5-like Fatty Acyl Desaturase from the Cephalopod Octopus vulgaris (Cuvier 1797) Involved in the Biosynthesis of Essential Fatty Acids

Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast’s endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C18 PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.