Long-chain polyunsaturated fatty acid biosynthesis in a land-crab with advanced terrestrial adaptations: Molecular cloning and functional characterization of two fatty acyl elongases.

Depending on the presence and activities of the front-end fatty acyl desaturases and elongation of very long-chain fatty acid (Elovl) enzymes, animals have different capacities for long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis. Successful land colonisation in brachyuran crabs requires a shift towards terrestrial food chain with limited LC-PUFA availability. We cloned and functionally characterised two elovl genes from the purple land crab Gecarcoidea lalandii. The two Elovl contained all the necessary motifs of a typical polyunsaturated fatty acids (PUFA) Elovl and phylogenetically clustered in the Elovl1 and Elovl6 clades, respectively. The G. lalandii Elovl1 elongated saturated fatty acids, with low activities towards C20 and C22 PUFA substrates. Moreover, the G. lalandii Elovl6 was particularly active in the elongation of C18 PUFA, although it also recognised monounsaturated fatty acids as substrates for elongation. Collectively, the herein characterised G. lalandii elovl paralogues fulfil all the elongation steps involved in the LC-PUFA biosynthetic pathways. Tissue distribution of the G. lalandii elovl genes, along with the FA composition analyses, suggest the hepatopancreas and gill as key metabolic sites for fatty acid elongation. However, current data suggest that G. lalandii is unable to rely solely on biosynthesis to fulfil LC-PUFA requirements, since front-end desaturase appears to be absent in this species and other decapods.

Two Elongases, Elovl4 and Elovl6, Fulfill the Elongation Routes of the LC-PUFA Biosynthesis Pathway in the Orange Mud Crab (Scylla olivacea).

While the capacity for long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis has been elucidated in vertebrates and several invertebrate phyla, the comparative knowledge in crustaceans remains vague. A key obstacle in mapping the full spectrum of LC-PUFA biosynthesis in crustacean is the limited evidence of the functional activities of enzymes involved in desaturation or elongation of polyunsaturated fatty acid substrates. In this present study, we report on the cloning and functional characterization of two Elovl elongases from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis suggest these two Elovl as putative Elovl4 and Elovl6, respectively. Using the recombinant expression system in Saccharomyces cerevisiae, we demonstrate the elongation capacity for C18-C22 PUFA substrates in the S. olivacea Elovl4. The S. olivacea Elovl6 elongated saturated fatty acids, monounsaturated fatty acids, and interestingly, C18-C20 PUFA. Taken together, both Elovl fulfill the elongation steps required for conversion of C18 PUFA to their respective LC-PUFA products. Elovl4 is expressed mainly in the hepatopancreas and gill tissues, while Elovl6 is predominant in digestive tissues. The mRNA expression of both enzymes was higher in mud crabs fed with vegetable oil-based diets. Tissue fatty acid composition also showed the existence of LC-PUFA biosynthesis intermediate products in tissues expressing these two elongases. In summary, we report here two novel Elovl with PUFA elongating activities in a marine brachyuran. This will contribute significantly to the understanding of the LC-PUFA biosynthesis pathway in crustaceans and advance the development of aquafeed for intensive farming of the mud crab.

Molecular cloning, phylogenetic analysis and functional characterisation of an Elovl7-like elongase from a marine crustacean, the orange mud crab (Scylla olivacea).

The capacity of crustaceans to biosynthesise long-chain polyunsaturated fatty acids has yet to be fully defined, due to the lack of evidence on the functional activities of enzymes involved in desaturation or elongation of fatty acid substrates. We report here the cloning and in vitro functional analysis of an elongase from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis placed the elovl close to the vertebrate Elovl1 and Elovl7 clade, which is distinct from the other remaining five Elovl families. The elongase was also clustered together with several elongases from crustaceans and insects. This elongase showed activities towards 16:1n-7, and at lower rate, linoleic acid (18:2n-6) and linolenic acid (18:3n-3). To our knowledge this is the first description of a functional enzyme involved in biosynthesis of long-chained polyunsaturated fatty acids in a crustacean species. Expression of the S. olivacea elovl7-like mRNA was prominent in stomach, intestine and gill tissues, due to the need to regulate the permeability of epithelial tissue through modification of fatty acid compositions. The implication of our findings, in terms of ability of Crustacea phylum to biosynthesise polyunsaturated fatty acids is discussed.