Biosynthesis of Long-Chain Polyunsaturated Fatty Acids in Marine Gammarids: Molecular Cloning and Functional Characterisation of Three Fatty Acyl Elongases

Abstract

Long-chain (C20–24) polyunsaturated fatty acids (LC-PUFAs) are essential nutrients that are mostly produced in marine ecosystems. Previous studies suggested that gammarids have some capacity to endogenously produce LC-PUFAs. This study aimed to investigate the repertoire and functions of elongation of very long-chain fatty acid (Elovl) proteins in gammarids. Our results show that gammarids have, at least, three distinct elovl genes with putative roles in LC-PUFA biosynthesis. Phylogenetics allowed us to classify two elongases as Elovl4 and Elovl6, as they were bona fide orthologues of vertebrate Elovl4 and Elovl6. Moreover, a third elongase was named as “Elovl1/7-like” since it grouped closely to the Elovl1 and Elovl7 found in vertebrates. Molecular analysis of the deduced protein sequences indicated that the gammarid Elovl4 and Elovl1/7-like were indeed polyunsaturated fatty acid (PUFA) elongases, whereas Elovl6 had molecular features typically found in non-PUFA elongases. This was partly confirmed in the functional assays performed on the marine gammarid Echinogammarus marinus Elovl, which showed that both Elovl4 and Elovl1/7-like elongated PUFA substrates ranging from C18 to C22. E. marinus Elovl6 was only able to elongate C18 PUFA substrates, suggesting that this enzyme does not play major roles in the LC-PUFA biosynthesis of gammarids.

Functional characterization reveals a diverse array of metazoan fatty acid biosynthesis genes

“…having a rather general elongation capacity similar to the T. californicus orthologue. Five P. littoralis elongases (elovl1a-e) clustered within the recently discovered Pancrustacea-specific elovl1/7-like clade(Ribes-Navarro et al, 2021), and the detected strong elongation capacities of the tested elongases further emphasize the importance of this clade. The results from P. littoralis elovl1c and elovl1e as well as T. californicus "elo2" show that elongases with a "QXXHH" histidine box instead of the usual "HXXHH" histidine box can still exhibit elongation capacities.…”

“…While PUFA elongases have been successfully functionally characterized in other crustacean lineages such as decapods, branchiopods and amphipods (Mah et al, 2019; Ribes‐Navarro et al, 2021; Sun et al, 2020; Ting et al, 2020), the occurrence and functionality of methyl‐end and front‐end desaturases in these lineages remains highly questionable (Chen et al, 2017; Kabeya et al, 2021; Lin et al, 2017; Monroig & Kabeya, 2018; Nielsen et al, 2019; Ting et al, 2021; Wu et al, 2018; Yang et al, 2013). In our phylogenetic study, we detected a putative front‐end desaturase in a transcriptome of the decapod Eurypanopeus depressus clustering together with the T. californicus Δ5 front‐end desaturase “ Fed5 ” with high bootstrap support (86%).…”

Effects of diet and temperature on the fatty acid composition of the gammarid Gammarus locusta fed alternative terrestrial feeds

“…PUFA such as EPA and DHA from dietary FA (Castro et al, 2016;Monroig and Kabeya, 2018;Monroig et al, 2022). A recent report on the three different elongases in the marine gammarid E. marinus (Ribes-Navarro et al, 2021) indicates the potential capacity for endogenous production of LC-PUFA in marine gammarids, but the presence and activity of desaturases remain to be elucidated. The presence of EPA and DHA can be due to reasons other than endogenous biosynthesis, such as the accumulation and retention of these FA from sources other than the diet provided, like the occasional and practically unavoidable presence of prey organisms such as copepods and rotifers in the culture tanks during the experiment.…”

Examination of gammarid transcriptomes reveals a widespread occurrence of key metabolic genes from epibiont bdelloid rotifers in freshwater species

“…The pYES2 constructs containing the ORF of the genes of interest ( pYES2-Elovl25, pYES2-Fed1, pYES2-Fed2, pYES2Fed3, pYES2-Fed4, pYES2-ωx1 and pYES2-ωx2) were individually transformed into INVSc1 S. cerevisiae competent cells (Thermo Fisher Scientific) using the S.c. EasyComp yeast transformation kit (Thermo Fisher Scientific). Selection and growth of recombinant yeast were carried out as described by Ribes-Navarro et al [44]. To test the capacity of the elongase and desaturase enzymes, recombinant yeast expressing one sole LC-PUFA biosynthesis gene was supplemented with one of the following potential PUFA substrates selected according to LC-PUFA biosynthetic pathways (figure 1).…”