Production of eicosapentaenoic acid by application of a delta-6 desaturase with the highest ALA catalytic activity in algae

Abstract

Abstract:Dunaliella salina is a unicellular green alga with a high α-linolenic acid (ALA) level, but a low eicosapentaenoic acid (EPA) level. In a previous analysis of the catalytic activity of delta 6 fatty acid desaturase (FADS6) from various species, FADS6 from Thalassiosira pseudonana (TpFADS6), a marine diatom, showed the highest catalytic activity for ALA. In this study, to enhance EPA production in D. salina, FADS6 from D. salina (DsFADS6) was identified, and substrate specificities for DsFADS6 and TpFADS6 were characterized. Furthermore, a plasmid harboring the TpFADS6 gene was constructed and overexpressed in D. salina. Our results revealed that EPA production reached 21.3 ± 1.5 mg/L in D. salina transformants. To further increase EPA production, myoinositol (MI) was used as a growth-promoting agent; it increased the dry cell weight of D. salina transformants, and EPA production reached 91.3 ± 11.6 mg/L. The combination of 12% CO 2 aeration with glucose/KNO 3 in the medium improved EPA production to 192.9 ± 25.7 mg/L in the Ds-TpFADS6 transformant. We confirmed that the increase in ALA was optimal at 8 °C; the EPA percentage reached 41.12 ± 4.78%. The EPA yield was further increased to 554.3 ± 95.6 mg/L by supplementation with 4 g/L perilla seed meal (PeSM), 500 mg/L MI, and 12% CO 2 aeration with glucose/KNO 3 at varying temperatures. EPA production and the percentage of EPA in D. salina were 343.8-fold and 25-fold higher than those in wild-type D. salina, respectively. Importance: FADS6 from Thalassiosira pseudonana, which demonstrates high catalytic activity toward α-linolenic acid, was used to enhance EPA production by Dunaliella salina. Transformation of FADS6 from Thalassiosira pseudonana into Dunaliella salina with myoinositol, CO 2 , low temperatures, and perilla seed meal supplementation substantially increased EPA production in Dunaliella salina to 554.3 ± 95.6 mg/L. Accordingly, D. salina could be a potential alternative source of EPA and is suitable for its large-scale production.

Evidence of Interdomain Ammonium Cross-Feeding From Methylamine- and Glycine Betaine-Degrading Rhodobacteraceae to Diatoms as a Widespread Interaction in the Marine Phycosphere

“…The addition of glycolate caused a doubling of OD 600 compared to succinate-only control cultures, while the addition of formate and glyoxylate had no effect. The common algal exudates GBT and DMSP could also be used as growth substrates, but the polyunsaturated fatty acid EPA, which is produced by diatoms and other microalgae ( Good et al, 2015 ; Shi et al, 2018 ), could apparently not be used as a growth substrate and even had negative effects on the growth of strain KarMa with glucose (not shown). The polyamines putrescine and spermidine are also diatom-related DON compounds, as they occur in the ornithine–urea cycle and are involved in precipitation of the silicified cell wall ( Kröger et al, 2000 ; Allen et al, 2006 ; Sumper and Lehmann, 2006 ).…”

Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

“…Then, the expression level of each chimeric desaturase in Pichia pastoris was determined by western blotting analysis as described previously ( Shi et al, 2015); each sample loading volume was adjusted to the same level according to the western blot results to determine desaturase activity of each chimera. Chimeric desaturase activity were analyzed by gas chromatography (GC) as described previously ( Shi et al, 2016; Shi et al, 2018b).…”

Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

“…Approximately 0.5 g (dry weight) of G. candidum cells was ground to a fine powder with a precooled mortar and pestle using liquid nitrogen. Total RNA was isolated using the RNAprep Pure Plant Kit, and reverse‐transcribed with QuantScript RT Kit as described previously . RT‐qPCR was performed as described previously , and the transcript levels were calculated using the method.…”

“…In EFA biosynthesis, Δ12 fatty acid desaturase (FADS12), Δ15 fatty acid desaturase (FADS15) and Δ6 fatty acid desaturase (FADS6) play a key role in regulating the level of EFAs. In a previous study, the molecular mechanism, substrate specificity and catalytic activity for FADS6 were analyzed and applied to PUFA synthesis . FADS12 is a key bifunctional membrane‐bound desaturase that converts oleic acid (OA, 18:1 Δ9 ) to linoleic acid (LA, 18:2 Δ9,12 ) and LA to α‐linolenic acid (ALA, 18:3 Δ9,12,15 ) by introducing a double bond between the carbons 12 and 13 from the carboxyl end of the substrate in the biosynthesis of EFAs .…”