Introduction
The rapid growth of aquaculture has prompted the exploration of sustainably sourced ingredients that, like traditional marine ingredients as fishmeal and fish oil , are rich in the essential long-chain (≥C20 ) polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic acid (EPA, 20:5n-3) , arachidonic acid (ARA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3) . Biomasses derived from aquatic invertebrates, particularly polychaetes, have emerged as promising LC-PUFA-rich ingredients for aquafeeds [1]. Many aquatic invertebrates have the capacity for endogenous production (biosynthesis) of LC-PUFAs, with complete sets of key enzymes termed methyl-end (or “omega”) desaturases (ωx), front-end desaturases (Fed), and elongases (Elovl) [2]. A unique trait of the LC-PUFA biosynthetic pathways from invertebrates including polychaetes, is the occurrence of ωx, enzymes that enable the de novo biosynthesis of PUFAs and, from them, n-3 LC-PUFAs [3]. This metabolic capacity, combined with their detritivorous feeding habits, makes polychaetes ideal candidates for Circular Economy approaches, where low-value, n-3 LC-PUFA-poor side streams from bioindustries can be recycled into high-value, n-3 LC-PUFA-rich biomass. The aim of the present study was to report on results collected from the molecular and functional characterisation of the LC-PUFA biosynthesising desaturases and elongases from t he nereid polychaete Hediste diversicolor . Furthermore, due to the capacity of polychaetes to modulate the LC-PUFA biosynthesis in response to environmental factors , we carried out an in vivo feeding trial aiming to assess the effects of temperature and salinity on LC-PUFA biosynthesis in H. diversicolor juveniles fed on diets composed of side streams from the aquaculture and biogas industries.
Materials and Methods
The molecular characterisation of desaturases and elongases involved in the H. diversicolor LC-PUFA was conducted by determining the phylogeny of the encoded enzymes. Searches for relevant sequences were performed in both public and non-public genomic/transcriptomic databases. Functional characterisation of the H. diversicolor desaturases and elongases was done by heterologous expression of their coding regions in brewer’s yeast (Saccharomyces cerevisiae). Conversion of the enzymes was determined by analysing the proportion of the fatty acid substrate converted to the corresponding product(s). To evaluate the influence of temperature and salinity on LC-PUFA biosynthesis, an in vivo feeding trial was conducted using H. diversicolor reared on side stream-based diets. Polychaetes were cultured for 28 days along a 5-step temperature and salinity gradient ranging from 7.7 to 17.9 °C and 5 to 40 psu, respectively. Growth, survival and fatty acid (FA) profiles of the worms were analysed.
Results and Discussion
Molecular and functional analyses revealed that H. diversicolor possess three elongases (Elovl2/5, Elovl4 and Elovl1/7) capable of elongating C18 C20 and C22 PUFA substrates , and two front-end desaturases (Fed1 and Fed2), with Δ5 and Δ6/Δ8 desaturase activity , respectively . This enzymatic capacity is shared with other invertebrates such as molluscs, echinoderms and annelids [2]. Collectively, these enzymes, along with the two methyl-end desaturases reported by [4] , confirm that H. diversicolor has complete pathways to biosynthesise LC-PUFAs , including ARA and EPA (Fig. 1). However, the data collected suggest that H. diversicolor is unable to biosynthesise DH A. Regarding the regulation of LC-PUFA biosynthesis by temperature and salinity, our findings showed tha t the FA composition of H. diversicolor remains relatively stable, regardless of the temperature conditions . Nevertheless, low salinity (5 and 15 psu) was associated with increased levels of n-3 LC-PUFAs primarily driven by EPA, in the lipids of the polychaetes . These results support that the activities of the LC-PUFA biosynthetic enzymes (desaturases and elongases) characterised in y east, appear to be operative in vivo according to the high levels of LC-PUFAs found in the lipids of H. diversicolor fed on diets with very limited LC-PUFAs. Overall, our findings underscore the biotechnological potential of H. diversicolor for generating high nutritional value biomass for aquaculture, following Circular Economy principles.
Acknowledgements
IMPROMEGA RTI2018-095119-B-I00 financed by MCIN/ AEI /10.13039/501100011033/ and by FEDER A way to make Europe. SIDESTREAM (Grant 68) co-financed by MCIN/AEI /10.13039/501100011033 and by EU NextGenerationEU/PRTR. Project THINKINAZUL/2021/26, funded by MICIU, EU NextGenerationEU and by Generalitat Valenciana.
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