Characterization, mRNA expression and nutritional regulation of a Δ6 fatty acyl desaturase-like gene by dietary DHA levels of Perinereis aibuhitensis (Gube, 1878)

The Δ6 fatty acyl desaturase (Δ6 Fad) enzyme plays an important role in the pathways for the biosynthesis of the highly unsaturated fatty acids (HUFA) by converting linoleic (LA, 18:2n-6) and α-linolenic (LNA, 18:3n-3) acids to 18:3n-6 and 18:4n-3, respectively. In the present study, a Δ6 Fad-like gene was cloned and its characterisation, tissue expression and nutritional regulation by dietary DHA (docosahexaenoic acid) levels were analysed in the clamworm Perinereis aibuhitensis. The full-length cDNA was 1535 bp including a 1335 bp open reading frame (ORF) encoding a polypeptide of 444 amino acids, a 113 bp 50’-UTR and an 87 bp 3’-UTR with a poly (A) tail. Sequence comparison revealed that the predicted protein shared higher homologies with Δ6 Fad from Apostichopus japonicus with 56% identify. The tissue distribution of P. aibuhitensis Δ6 Fad-like mRNA was found predominantly in intestine, followed by head, esophagus, stomach and other tissues. Quantitative real-time PCR showed that the Δ6 Fad-like transcriptional levels in intestine gradually decreased with increased content of dietary DHA.

Transcriptional regulation mechanism of sterol regulatory element binding proteins on Δ6 fatty acyl desaturase in razor clam Sinonovacula constricta

The razor clam, Sinonovacula constricta, contains high levels of long-chain PUFA (LC-PUFA), which are critical for human health. In addition, S. constricta is the first marine mollusc demonstrated to possess Δ6 fatty acyl desaturase (Fad) and complete LC-PUFA biosynthetic ability, providing a good representative to investigate the molecular mechanism of sterol regulatory element binding proteins (SREBP) in regulating Δ6 Fad for LC-PUFA biosynthesis in marine molluscs. Herein, S. constricta SREBP and Δ6 Fad promoter were cloned and characterised. Subsequently, dual luciferase and electrophoretic mobility shift assays were conducted to explore the SREBP binding elements in the core regulatory region of S. constricta Δ6 Fad promoter. Results showed that S. constricta SREBP had a very conservative basic helix-loop-helix-leucine zipper motif, while S. constricta Δ6 Fad promoter exhibited very poor identity with teleost Fads2 promoters, indicating their differentiation during evolution. A 454 bp region harbouring a core sequence in S. constricta Δ6 Fad promoter was predicted to be essential for the transcriptional activation by SREBP. This was the first report on the regulatory mechanism of LC-PUFA biosynthesis in marine molluscs, which would facilitate optimising the LC-PUFA biosynthetic pathway of bivalves in further studies.

Functional characterization of fatty acyl desaturase Fads2 and Elovl5 elongase in the Boddart’s goggle-eyed goby, Boleophthalmus boddarti (Gobiidae) suggest an incapacity for long-chain polyunsaturated fatty acid biosynthesis

Long-chain polyunsaturated fatty acid biosynthesis, a process to convert C18 polyunsaturated fatty acids to eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or arachidonic acid (ARA) requires the concerted activities of two enzymes, the fatty acyl desaturase (Fads) and elongase (Elovl). This study highlights the cloning, functional characterisation and tissue expression pattern of a Fads and Elovl from the Boddart’s goggle-eyed goby (Boleophthalmus boddarti), a mudskipper species widely distributed in the Indo-Pacific region. Phylogenetic analysis revealed that the cloned Fads and Elovl are clustered with other teleost Fads2 and Elovl5 orthologs, respectively. Interrogation of the genome of several mudskipper species, namely B. pectinirostris, Periophthalmus schlosseri and P. magnuspinnatus revealed a single Fads2 for each respective species while two elongases, Elovl5 and Elovl4 were detected. Using a heterologous yeast assay, the B. boddarti Fads2 was shown to possess low desaturation activity on C18 PUFA. In addition, there was no desaturation of C20 and C22 substrates. In comparison, the Elovl5 showed a wide range of substrate specificity, with capacity to elongate C18, C20 and C22 PUFA substrates. We identified an amino acid residue in the B. boddarti Elovl5 that affect the capacity to bind C22 PUFA substrate. Both genes are highly expressed in brain tissue. Among all tissues, DHA is highly concentrated in neuron-rich tissues while EPA is highly deposited in gills. Taken together, the results showed that due to disability of desaturation steps, B. boddarti is unable to biosynthesis LC-PUFA, relying on dietary intake to acquire these nutrients.