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% CO2 aeration with glucose/KNO3 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% CO2 aeration with glucose/KNO3 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.ImportanceFADS6 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, CO2, 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.
Highlights
Recent clinical and epidemiological studies have indicated that polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5Δ5, 8, 11, 14, 17), are essential nutrients and play crucial roles in the treatment of various human diseases [1,2,3], such as neuropsychiatric disorders [4], rheumatoid arthritis [5], inflammatory diseases [6, 7], hypertension [8],and cardiovascular diseases [9]
Homology was highest near the HPGG domain and in the three conserved HIS-rich domains (Fig. 2). These results suggest that DsFADS6 may encode a delta 6 desaturase involved in the biosynthesis of γ-linolenic acid (GLA) and stearidonic acid (SDA) in D. salina
Substrate concentration has no effect on catalytic activity in yeast expressing DsFADS6; we found that there was no substrate specificity for TpFADS6, irrespective of fatty acid concentration
Summary
Recent clinical and epidemiological studies have indicated that polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5Δ5, 8, 11, 14, 17), are essential nutrients and play crucial roles in the treatment of various human diseases [1,2,3], such as neuropsychiatric disorders [4], rheumatoid arthritis [5], inflammatory diseases [6, 7], hypertension [8],and cardiovascular diseases [9]. Many recent studies have developed technologies to accumulate EPA directly from various microalgae [11]. Among Chlorophyceae, Dunaliella salina (D. salina) is a unicellular wall-less alga that produces β-carotene and has a high tolerance to salt, temperature and light. This alga is quite easy to cultivate and has a relatively high lipid content, especially α-linolenic acid (ALA, 18:3Δ9,12,15).
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