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

Abstract

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".

Fig 1. Biosynthetic pathways of LC-PUFA from C₁₈ PUFA precursors accepted in vertebrates.

Reactions catalysed by fatty acyl desaturases are designated as “Δx” (Δ6, Δ5, Δ4 and Δ8), whereas elongation reactions are indicated as “elongase”.

Fig 2. Comparison of the deduced amino acid sequence of the Paracentrotus lividus desaturases with those of mice (Mus musculus), zebrafish (Danio rerio) and common octopus (Octopus vulgaris).

Identical sequences are shaded black. All the Fads sequence from P. lividus contained typically conserved regions in members of the front-end desaturase family, including three histidine boxes (HXXXH, HXXHH and QXXHH), a putative cytochrome b5-like region (marked as a dotted line) and a heme-binding motif (HPGG).

Fig 3. Phylogenetic tree comparing the deduced amino acid (aa) sequence of the Paracentrotus lividus fatty acyl desaturases (Fads) with other Fads-like sequences from different organisms (vertebrates and invertebrates).

FadsA, FadsC1 and FadsC2 are highlighted in black. The tree was constructed using the Neighbour Joining method [38]. The horizontal branch length is proportional to aa substitution rate per site. The numbers represent the frequencies (%) with which the tree topology presented was replicated after 1,000 iterations. Asterisks denote deduced aa sequences derived from transcriptome shotgun assembly (TSA) from several echinoderm species.

Fig 4. Functional characterisation of FadsA from Paracentrotus lividus in transformed yeast Saccharomyces cerevisiae.

Additional 18:1n-13 peak was observed in all the yeast transformed with pYES2-FadsA (B–F) compared with the yeast transformed with empty pYES2 vector (A). The yeast transformed with pYES2-FadsA were also grown in the presence of fatty acid (FA) substrates (indicated by asterisk), namely 20:3n-3 (C), 20:2n-6 (D), 20:4n-3 (E) and 20:3n-6 (F). Desaturation products are indicated accordingly in each panel. Peaks 1–4 in all panels are the main endogenous FA of S. cerevisiae, namely 16:0 (1), 16:1 isomers (2), 18:0 (3), 18:1n-9 (4). Vertical axis, FID response; horizontal axis, retention time.

Fig 5. Functional characterisation of FadsC1 and FadsC2 from Paracentrotus lividus in transformed yeast S. cerevisiae.

The fatty acid (FA) profiles of yeast transformed with pYES2-FadsC1 (A, B) and pYES2-FadsC2 (C, D) were determined after they were grown in the presence of FA substrates (indicated by asterisk), namely 20:3n-3 (A, C) and 20:2n-6 (B, D). Moreover, desaturation products are indicated accordingly in each panel. Peaks 1–4 in all panels are the main endogenous FA of S. cerevisiae, namely 16:0 (1), 16:1 isomers (2), 18:0 (3), 18:1n-9 (4). Vertical axis, FID response; horizontal axis, retention time.