Using Glycerol to Produce European Sea Bass Feed With Oleaginous Microbial Biomass: Effects on Growth Performance, Filet Fatty Acid Profile, and FADS2 Gene Expression

Using a circular economy concept, the present study investigated the use of crude glycerol, a primary by-product of biodiesel production, as a low-priced nutrient source for heterotrophic cultivation of the fungus-like protist Schizochytrium limacinum SR21 strain. The whole biomass of this oleaginous microorganism, rich in docosahexaenoic acid (DHA) and high-quality proteins, was then paired with a vegetable oil (VO) source and used to replace fish oil (FO) in European sea bass (Dicentrarchus labrax) feeds. Four nutritionally balanced diets were formulated: diet FO (a FO-based diet), diet VO + 0 (a VO-based diet without S. limacinum), and diets VO + 5 and VO + 10 that were VO-based feeds supplemented with 5 and 10% of S. limacinum, respectively. After a 3-month feeding trial, fish of all dietary groups tripled their initial weight, but growth and feeding efficiencies of D. labrax were not significantly different among treatments. Although the formulated diets were balanced for polyunsaturated fatty acids (PUFAs), fish fed with feeds containing either VO or VO plus 5 and 10% of S. limacinum biomass had significantly higher levels of PUFAs in the flesh than fish fed the FO-based diet. Values of health-related lipid indexes, such as atherogenicity index, thrombogenicity index, and flesh lipid quality as well as n-6/n-3 and PUFAs/SFAs ratios confirmed the high nutritional value of sea bass filet, thus representing a healthy product for human consumption. Although the PUFAs/SFAs ratio showed a significantly higher value in fish fed with VO-based diets supplemented with S. limacinum than in those fed with FO diet, suggesting a better filet quality, the n-6/n-3 ratio clearly indicated that filet quality of dietary group FO was best (value of 0.55) and that of group VO + 10 second best (value of 0.98). We also evaluated the nutritional regulation of Δ6-desaturase (or fads2) gene expression in European sea bass liver. European sea bass fed the VO + 0 diet had the highest number of mRNA copies for Δ6-desaturase (or fads2), fish fed with diet VO + 10 the lowest. Our study adds to the growing body of literature concerning the use of thraustochytrid biomass as a valid alternative to marine-derived raw materials for European sea bass feeds.

Synthesis of DHA (omega-3 fatty acid): FADS2 gene polymorphisms and regulation by PPARα

In humans, in several biological systems, in particular the nervous system, the FADS2 gene transcribes Δ6-desaturase, which is the rate-limiting enzyme for converting α-linolenic acid into docosahexaenoic acid (an n-3 fatty acid). The peroxisome proliferator-activated receptor α (PPARα) modulates the transcription of FADS2 gene by interacting with a second transcription factor: the retinoid X receptor α (RXRα). These transcription factors take the form of a PPARα-RXRα heterodimer and are modulated by the ligands that modify their respective structures and enable them to bind to the peroxisome proliferator response element (PPRE) located in the promoter region of the FADS2 gene. Free estradiol induces the activation of PPARα via two pathways (i) transcription through genomic action mediated by an estrogen receptor; (ii) a non-genomic effect that allows for phosphorylation and activates PPARα via the ERK1/2-MAPK pathway. Phosphorylation is an on/off switch for PPARα transcription activity. Since Δ6-desaturase expression is retro-inhibited by free intracellular DHA in a dose-dependent manner, this position paper proposes an original hypothesis: if DHA simultaneously binds to both phosphorylated PPARα and RXRα, the resulting DHA-PPARαP-RXRα-DHA heterodimer represses FADS2 gene via PPRE. The retinoic acids-RARα-RXRα-DHA heterodimer would not dissociate from corepressors and would prevent coactivators from binding to FADS2. We speculate that SNPs, which are mostly located on PPRE, modulate the binding affinities of DHA-PPARαP-RXRα-DHA heterodimer to PPRE. The DHA-PPARαP-RXRα-DHA heterodimer’s greater affinity for PPRE results in a decreased production of D6D and DHA. FADS2 promoter polymorphism would increase the competition between DHA and other ligands, in accordance with their concentrations and affinities.

Juniperonic Acid Biosynthesis is Essential in Caenorhabditis elegans Lacking Δ6 Desaturase (fat-3) and Generates New ω-3 Endocannabinoids

In eukaryotes, the C20:4 polyunsaturated fatty acid arachidonic acid (AA) plays important roles as a phospholipid component, signaling molecule and precursor of the endocannabinoid-prostanoid axis. Accordingly, the absence of AA causes detrimental effects. Here, compensatory mechanisms involved in AA deficiency in Caenorhabditis elegans were investigated. We show that the ω-3 C20:4 polyunsaturated fatty acid juniperonic acid (JuA) is generated in the C. elegans fat-3(wa22) mutant, which lacks Δ6 desaturase activity and cannot generate AA and ω-3 AA. JuA partially rescued the loss of function of AA in growth and development. Additionally, we observed that supplementation of AA and ω-3 AA modulates lifespan of fat-3(wa22) mutants. We described a feasible biosynthetic pathway that leads to the generation of JuA from α-linoleic acid (ALA) via elongases ELO-1/2 and Δ5 desaturase which is rate-limiting. Employing liquid chromatography mass spectrometry (LC-MS/MS), we identified endocannabinoid-like ethanolamine and glycerol derivatives of JuA and ω-3 AA. Like classical endocannabinoids, these lipids exhibited binding interactions with NPR-32, a G protein coupled receptor (GPCR) shown to act as endocannabinoid receptor in C. elegans. Our study suggests that the eicosatetraenoic acids AA, ω-3 AA and JuA share similar biological functions. This biosynthetic plasticity of eicosatetraenoic acids observed in C. elegans uncovers a possible biological role of JuA and associated ω-3 endocannabinoids in Δ6 desaturase deficiencies, highlighting the importance of ALA.

Plastidic Δ6 fatty-acid desaturases with distinctive substrate specificity regulate the pool of c18-pufas in the ancestral picoalga ostreococcus tauri

ABSTRACTEukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several microalgae, including O. tauri, plastidic C18-PUFA are specifically regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. Sequence retrieval from O. tauri desaturases, highlighted two putative Δ6/Δ8-desaturases sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 desaturases. Their overexpression in heterologous hosts, including N. benthamiana and Synechocystis, unveiled their Δ6-desaturation activity and plastid localization. O. tauri lines overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount under phosphate starvation but didn’t show any obvious physiological alterations. Detailed lipid analyses from the various overexpressing hosts, unravelled that the substrate features involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native host. The distribution of plastidial Δ6-desaturase products in eukaryotic hosts suggested the occurrence of C18-PUFA export from the plastid.One sentence summaryOsteococcus tauri plastidic lipid C18-PUFA remodelling involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for both head-group and acyl-chain.

Consensus mutagenesis and ancestral reconstruction provide insight into the substrate specificity and evolution of the front-end Δ6-desaturase family

ABSTRACTMarine algae are a major source of omega (ω)-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae begins with the desaturation of fatty acids by Δ6-desaturases and enzymes from different species display a range of specificities towards ω3 and ω6 LCPUFAs. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This dataset provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.