Cloning, expression and activity analysis of homologous genes of diacylglycerol acyltransferase 2 in Mortierella alpina

Abstract:

[Background] Mortierella alpina is an oleaginous fungus that can accumulate a large amount of arachidonic acid (AA). The fatty acids produced by M. alpina are mainly assembled on the glycerol skeleton and present in the form of triacylglycerol (TAG). Diacylglycerol acyltransferase (DGAT) is a key enzyme in the biosynthetic pathway of TAG and plays an important role in TAG production of M. alpina. [Objective] This study explored the functional characteristics of M. alpina DGAT2 in TAG biosynthesis, aiming to provide a reference for promoting the TAG production of oleaginous fungi and improving the fatty acid composition of TAG. [Methods] Two candidate genes MaDGAT2A/2B were screened from the M. alpina ATCC32222 genome by sequence alignment. Functional characterization of MaDGAT2A/2B were performed by heterologous expression in Saccharomyces cerevisiae. Then the activity of MaDGAT2A/2B was further analyzed by detecting TAG yield under the condition of exogenous addition of AA. Finally, the in vivo activity of MaDGAT2A/2B was analyzed by detecting the total fatty acid yield and composition of the M. alpina recombinant strains overexpressing MaDGAT2A/2B. [Results] The transforming with MaDGAT2A gene could increase the production of TAG in S. cerevisiae to 3.06% of dry cell weight, which was 4.91-fold of the control group; while MaDGAT2B did not significantly increase the yield of TAG in S. cerevisiae. When AA was added exogenously, MaDGAT2A/2B could promote TAG synthesis in the yeast recombinant strains. The TAG content of recombinant yeast expressing MaDGAT2A was 3.67-fold of the control group. The TAG content of recombinant yeast expressing MaDGAT2B was 2.61-fold of the control group. Overexpression of MaDGAT2A/2B in M. alpina had no significant effect on the total fatty acid yield, but could significantly increase the content of AA in total fatty acids, up to 39.15%, an increase of 16.14% compared to the control group. [Conclusion] MaDGAT2A/2B could participate in the biosynthesis of TAG, indicating that the two proteins encoded by the two candidate genes have DGAT activity. And MaDGAT2A/2B could increase the content of AA in M. alpina, which is of great significance for improving the lipid composition of oleaginous fungi and increasing their application value.

Key words:diacylglycerol acyltransferase 2;Mortierella alpina;triacylglycerol;oleaginous fungus;arachidonic acid