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”.
Table 1.
Sequences of the primer pairs used in the molecular cloning and functional characterisation in yeast of the Paracentrotus lividus fatty acyl desaturases (FadsC1, FadsA and FadsC2).
Restriction sites for HindIII and XbaI are underlined (AAGCTT and TCTAGA, respectively).
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.
Table 2.
Substrate conversions of yeast Saccharomyces cerevisiae transformed with pYES2 containing the open reading frame (ORF) of the Paracentrotus lividus desaturases (FadsC1, FadsA and FadsC2).
Results are expressed as a percentage of total fatty acid (FA) substrate converted to desaturated products, with the corresponding activity (Δx) detected also shown. FA are designated using the ‘n-‘ nomenclature, except for the non-methylene interrupted FA produced from 20:3n-3 and 20:2n-6 where the ‘Δ’ nomenclature is used.
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.