椭圆小球藻高活性二酰基甘油酰基转移酶结构与功能的关系及其在微藻和高等植物中大幅合成三酰甘油的潜力探讨

Triacylglycerols (TAGs) are the most important storage lipids in microalgae. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final and committed step in TAG biosynthesis, and has been identified as the rate-limiting enzyme. Previous studies have shown that highly active DGAT can promote the synthesis of TAG. But there is still lack of highly active DGAT. In our previous work, we found that a DGAT (CeDGAT1) from Chlorella ellipsoidea has very high DGAT activity that can make the total lipid content of yeast strain INVSC1 increased by 140-200%, which is significantly higher by 23.52% than that made by the DGAT1 from Arabidopsis. In this study, the function of CeDGAT1 will be investigated in detail using yeast expression system; the amino acid residues crucial for the enzymatic activity of CeDGAT1 will be identified using site-directed and random mutagenesis, which can help us deeply understand the structure-function relationships of DGAT1, and get new DGAT1 with much higher activity. In addition, in order to identify its function and explore the possibility of mass production of TAG through expressing highly active DGAT1 in microalgae and higher plants, DGAT1 with high activity will be transformed into Chlorella, Arabidopsis and oilseed rape. In a word, this study would have important significance in clarifying the function of CeDGAT1 and woulb be helpful in understanding of mechanisms on lipid synthesis and accumulation in Chlorella. It will also provide a technical basis for improvement of oil content in microalgae and higher plants.

三酰甘油(TAG)是微藻的重要储藏物质。二酰基甘油酰基转移酶(DGAT)是合成TAG的限速酶。以往的研究表明高活性DGAT可以促进TAG大量合成。目前高活性DGAT缺乏。我们前期研究发现，来自椭圆小球藻的CeDGAT1具有很高的酶活性，能够使酵母的油脂含量提高140-200%, 比拟南芥菜的DGAT1高23.52%。本项目拟用酵母表达系统详细研究CeDGAT1的功能；利用定点和随机突变技术研究对CeDGAT1酶活性有重要影响的氨基酸残基，深刻探讨DGAT结构与功能的关系，获得编码更高活性DGAT1的基因；通过研究CeDGAT1表达谱及其在小球藻中的抑制表达及过表达，阐明CeDGAT1在小球藻油脂合成中的作用；在小球藻、拟南芥及油菜中过表达表达高活性DGAT1，探讨在微藻和高等植物中大量生产TAG的可能性。本项目对理解小球藻油脂合成和积累有重要意义，也能为微藻及高等植物油脂改良提供技术基础。

我们从椭圆小球中克隆一个高活性的DGAT1基因。在酵母中该基因可互补酵母突变体H1246合成TAG的能力，同时异源表达该基因在野生型酵母中可使其总脂肪酸含量提高140-200%，与高等植物的DGAT1相比具有更高的活性。通过定点突变分析，发现16个位点对CeDGAT1功能有着重要影响，其中Y553A位点的突变可使CeDGAT1活性提高6.7%。异源表达CeDGAT1可使拟南芥和油菜的种子油脂含量分别提提高8-37%和12-18%，对脂肪酸组分没有影响；粒重分别增加9-15% 和6-29%，且不会产生不利的农艺性状。为油料作物的油脂改良提供了重要的候选基因。