高山被孢霉中二酰甘油酰基转移酶2同源基因的克隆、表达和活性分析

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

摘要:

[背景] 高山被孢霉（Mortierella alpina）是一种可积累大量花生四烯酸（Arachidonic Acid，AA）的产油丝状真菌，其所产脂肪酸主要被组装到甘油骨架上以三酰甘油（Triacylglycerol，TAG）形式存在。二酰甘油酰基转移酶（Diacylglycerol Acyltransferase，DGAT）是TAG生物合成途径的关键酶，对于高山被孢霉TAG的生产具有重要意义。[目的] 通过探究高山被孢霉DGAT2在TAG生物合成方面的功能特点，以期为提高产油真菌的TAG产量及改善TAG的脂肪酸组成提供参考。[方法] 利用序列比对在高山被孢霉ATCC32222基因组中筛选出2个编码DGAT2的候选基因MaDGAT2A/2B，在酿酒酵母（Saccharomyces cerevisiae）中异源表达后进行功能分析，并在外源添加AA条件下通过检测TAG产量进一步分析MaDGAT2A/2B的活性，最后在高山被孢霉中同源过表达MaDGAT2A/2B，通过检测重组菌总脂肪酸产量及组分以分析MaDGAT2A/2B的体内活性。[结果] MaDGAT2A在S. cerevisiae中异源表达时，重组酵母菌TAG的产量达到细胞干重的3.06%，为对照组的4.91倍；而MaDGAT2B未明显提高重组酵母菌TAG的产量。在外源添加AA时，MaDGAT2A/2B均可显著促进重组酵母菌中TAG合成，表达MaDGAT2A的重组酵母菌TAG含量为对照组的3.67倍，表达MaDGAT2B的重组酵母菌TAG含量为对照组的2.61倍。MaDGAT2A/2B在高山被孢霉中过表达对其总脂肪酸产量无显著影响，但可显著提高总脂肪酸中AA的含量，AA占总脂肪酸比例最高达到39.15%，相比对照组提高16.14%。[结论] MaDGAT2A/2B可以参与TAG的生物合成，表明2个候选基因编码的蛋白具有DGAT活性，并且可提高高山被孢霉脂肪酸中AA的含量，对于改善产油真菌的脂肪酸组成从而提高其应用价值具有重要意义。

关键词:二酰甘油酰基转移酶2;高山被孢霉;三酰甘油;产油真菌;花生四烯酸

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