Unique fatty acid desaturase capacities uncovered in Hediste diversicolor illustrate the roles of aquatic invertebrates in trophic upgrading.

Omega-3 (ω3 or n-3) long-chain polyunsaturated fatty acids (PUFA), including eicosapentaenoic acid and docosahexaenoic acid (DHA), play physiologically important roles in vertebrates. These compounds have long been believed to have originated almost exclusively from aquatic (mostly marine) single-cell organisms. Yet, a recent study has discovered that many invertebrates possess a type of enzymes called methyl-end desaturases (ωx) that enables them to endogenously produce n-3 long-chain PUFA and could make a significant contribution to production of these compounds in the marine environment. Polychaetes are major components of benthic fauna and thus important to maintain a robust food web as a recycler of organic matter and a prey item for higher trophic level species like fish. In the present study, we investigated the ωx enzymes from the common ragworm, Hediste diversicolor, a common inhabitant in sedimentary littoral ecosystems of the North Atlantic. Functional assays of the H. diversicolorωx demonstrated unique desaturation capacities. An ω3 desaturase mediated the conversion of n-6 fatty acid substrates into their corresponding n-3 products including DHA. A further enzyme possessed unique regioselectivities combining both ω6 and ω3 desaturase activities. These results illustrate that the long-chain PUFA biosynthetic enzymatic machinery of aquatic invertebrates such as polychaetes is highly diverse and clarify that invertebrates can be major contributors to fatty acid trophic upgrading in aquatic food webs. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

Molecular and functional characterisation of a putative elovl4 gene and its expression in response to dietary fatty acid profile in Atlantic bluefin tuna (Thunnus thynnus).

Elongation of very long-chain fatty acid 4 (Elovl4) proteins are involved in the biosynthesis of very long-chain (>C24) fatty acids and in many teleost fish species they are key enzymes in the pathway for the production of docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Therefore, Elovl4 may be particularly important in Atlantic bluefin tuna (ABT; Thunnus thynnus) characterised by having high DHA to EPA ratios. The present study cloned and characterised both the function and expression of an elovl4 cDNA from ABT. The Elovl4 had an open reading frame of 915 base pairs encoding a putative protein of 304 amino acids. Alignment and phylogenetic analyses indicated that the Elovl4 isoform identified in the present study was an Elovl4b. Functional characterisation demonstrated that the Elovl4b enzyme had elongase activity towards all the polyunsaturated fatty acid (PUFA) substrates assayed. The ABT Elovl4b contributed to DHA biosynthesis by elongation of EPA and DPA to 24:5n-3, the latter being desaturated to 24:6n-3 by the action of fads2 (Δ6 desaturase). Additionally, the ABT Elovl4b has a role in the biosynthesis of very long-chain PUFA up to C34, compounds of key structural roles in neural tissues such as eye and brain, which had high levels of elovl4b transcripts. Surprisingly, while the relative expression of fads2, required for the production of DHA from EPA, was increased in liver of ABT fed a diet with reduced levels of EPA and DHA, expression of elovl4b was reduced. Results indicated that ABT has enzymes necessary for endogenous production of DHA from EPA and demonstrate that Elovl4b can effectively compensate for absence of Elovl2.

Biosynthesis of long-chain polyunsaturated fatty acids in the razor clam Sinonovacula constricta: Characterization of four fatty acyl elongases and a novel desaturase capacity.

As an unusual economically important aquaculture species, Sinonovacula constricta possesses high levels of long-chain polyunsaturated fatty acids (LC-PUFA). Previously, our group identified fatty acyl desaturases (Fad) with Δ5 and Δ6 activities in S. constricta, which was the first report of Δ6 Fad in a marine mollusc. Here, we further successfully characterize elongases of very long-chain fatty acids (Elovl) in this important bivalve species, including one Elovl2/5, two Elovl4 isoforms (a and b) and a novel Elovl (c) with Elovl4 activity. In addition, we also determined the desaturation activity of S. constricta Δ6 Fad toward 24:5n-3 to give 24:6n-3, a key intermediate in docosahexaenoic acid (DHA) biosynthesis. Therefore, S. constricta is the first marine mollusc reported to possess all Fad and Elovl activities required for LC-PUFA biosynthesis via the 'Sprecher pathway'. This finding greatly increases our understanding of LC-PUFA biosynthesis in marine molluscs. Phylogenetic analysis by interrogating six marine molluscan genomes, and previously functionally characterized Elovl and Fad from marine molluscs, suggested that DHA biosynthetic ability was limited to a few species, due to the general lack of Δ4 or Δ6 Fad in most molluscs.

Elongation of very Long-Chain (>C24) Fatty Acids in Clarias gariepinus: Cloning, Functional Characterization and Tissue Expression of elovl4 Elongases.

Elongation of very long-chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of very long-chain (>C24) saturated and polyunsaturated fatty acids (FA). Previous studies have shown that fish possess two different forms of Elovl4, termed Elovl4a and Elovl4b. The present study aimed to characterize both molecularly and functionally two elovl4 cDNA from the African catfish Clarias gariepinus. The results confirmed that C. gariepinus possessed two elovl4-like elongases with high homology to two previously characterized Elovl4 from Danio rerio, and thus they were termed accordingly as Elovl4a and Elovl4b. The C. gariepinus Elovl4a and Elovl4b have open reading frames (ORF) of 945 and 915 base pairs, respectively, encoding putative proteins of 314 and 304 amino acids, respectively. Functional characterization in yeast showed both Elovl4 enzymes have activity towards all the PUFA substrates assayed (18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3, 22:4n-6 and 22:6n-3), producing elongated products of up to C36. Moreover, the C. gariepinus Elovl4a and Elovl4b were able to elongate very long-chain saturated FA (VLC-SFA) as denoted by increased levels of 28:0 and longer FA in yeast transformed with elovl4 ORF compared to control yeast. These results confirmed that C. gariepinus Elovl4 play important roles in the biosynthesis of very long-chain FA. Tissue distribution analysis of elovl4 mRNAs showed both genes were widely expressed in all tissues analyzed, with high expression of elovl4a in pituitary and brain, whereas female gonad and pituitary had the highest expression levels for elovl4b.

Two alternative pathways for docosahexaenoic acid (DHA, 22:6n-3) biosynthesis are widespread among teleost fish.

Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called "Sprecher pathway", a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the "Δ4 pathway". In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts.

Biosynthesis of Polyunsaturated Fatty Acids in Sea Urchins: Molecular and Functional Characterisation of Three Fatty Acyl Desaturases from Paracentrotus lividus (Lamark 1816).

Sea urchins are broadly recognised as a delicacy and their quality as food for humans is highly influenced by their diet. Lipids in general and the long-chain polyunsaturated fatty acids (LC-PUFA) in particular, are essential nutrients that determine not only the nutritional value of sea urchins but also guarantee normal growth and reproduction in captivity. The contribution of endogenous production (biosynthesis) of LC-PUFA in sea urchins remained unknown. Using Paracentrotus lividus as our model species, we aimed to characterise both molecularly and functionally the repertoire of fatty acyl desaturases (Fads), key enzymes in the biosynthesis of LC-PUFA, in sea urchins. Three Fads, namely FadsA, FadsC1 and FadsC2, were characterised. The phylogenetic analyses suggested that the repertoire of Fads within the Echinodermata phylum varies among classes. On one hand, orthologues of the P. lividus FadsA were found in other echinoderm classes including starfishes, brittle stars and sea cucumbers, thus suggesting that this desaturase is virtually present in all echinoderms. Contrarily, the FadsC appears to be sea urchin-specific desaturase. Finally, a further desaturase termed as FadsB exists in starfishes, brittle stars and sea cucumbers, but appears to be missing in sea urchins. The functional characterisation of the P. lividus Fads confirmed that the FadsA was a Δ5 desaturase with activity towards saturated and polyunsaturated fatty acids (FA). Moreover, our experiments confirmed that FadsA plays a role in the biosynthesis of non-methylene interrupted FA, a group of compounds typically found in marine invertebrates. On the other hand, both FadsC desaturases from P. lividus showed Δ8 activity. The present results demonstrate that P. lividus possesses desaturases that account for all the desaturation reactions required to biosynthesis the physiological essential eicosapentaenoic and arachidonic acids through the so-called "Δ8 pathway".