以产油微藻海洋微拟球藻为模式的二酰甘油酰基转移酶功能机制研究

Certain wild microalgae are potentially capable of large-scale production of triacylglycerol (TAG), however dramatic improvement of their TAG productivity and quality is a prerequisite for their practical use in a cost-competitive microalgal biodiesel industry. Genetic engineering of oleaginous microalgae for enhanced oil productivity has been hindered by insufficient understanding of the mechanism of TAG synthesis in these non-model organisms. Diacylglycerol acyltransferase, which mediates the last and rate-limiting step of TAG synthesis, was considered to be able to control cellular TAG content. Recent studies show that Nannochloropsis oceanica IMET1 harbors thirteen DGAT homologs (NoDGAT) in its genome, the highest dose among any known organisms. Moreover temporal and spatial patterns of transcription have been highly distinct among the thirteen NoDGATs. Based on above-mentioned studies, this project proposes to characterize TAG synthetic activities of each of the thirteen NoDGATs in Saccharomyces cerevisiae neutral lipid-deficient quadruple knockout mutant H1246. Furthermore, individual physiological roles and the functional relationship among NoDGATs will be elucidated via in vitro and gene overexpression approaches. In the end, the first specifically functional mechanism among multiple DGATs in wild oleaginous microalgae will be established, and transgenic IMET1 strains with improved traits will be generated. These endeavors have implications in understanding and engineering of TAG metabolic pathways in Nannochloropsis spp. and should also lead to new insights on genetic improvement in microalgae.

当前一些野生微藻具备大规模养殖生产甘油三酯（TAG）的潜力，但其TAG合成效率和品质仍需大幅提升，才能满足微藻生物柴油产业的经济性要求。目前对微藻TAG代谢机制的了解尚不够深入，因而无法理性指导其遗传改良。研究表明，二酰甘油酰基转移酶（DGAT）负责催化TAG合成中的关键反应，调节其表达水平可改变胞内TAG含量。本课题组前期发现，海洋微拟球藻IMET1基因组中共有13个二酰甘油酰基转移酶同源基因（NoDGAT），为已知基因组物种中最多，而且各NoDGAT在时空表达上存在显著差异，暗示了各NoDGAT在功能上的差异性。基于上述研究，本项目计划通过酵母异源表达、体外酶学反应和内源过表达等手段，对13个NoDGAT的功能进行比较鉴定，进而首次阐明微藻中多个DGAT间的功能特异性机制。此工作对于探讨产油微藻的油脂代谢机理与性状改良具有重要意义。

当前一些野生微藻具备大规模养殖生产甘油三酯（TAG）的潜力，但其TAG合成效率和品质仍需大幅提升，才能满足微藻生物柴油产业的经济性要求。在本项目申请时，业界对微藻TAG代谢机制的了解尚不够深入，因而无法理性指导其遗传改良。前期研究表明，二酰甘油酰基转移酶（DGAT）负责催化TAG合成中的关键反应，调节其表达水平可改变胞内TAG含量。此外，海洋微拟球藻IMET1基因组中共有13个二酰甘油酰基转移酶同源基因（NoDGAT），而且各NoDGAT在时空表达上存在显著差异，暗示多NoDGAT在功能上的各异性。基于上述研究，本项目的主要研究内容为通过酵母异源表达、体外酶学反应和内源过表达等手段，对13个NoDGAT的功能进行比较鉴定，进而首次阐明微藻中多个DGAT间的功能特异性机制。经过近3年的研究工作，本项目的主要结果为：①在13个NoDGAT 拷贝中，5个NoDGAT被证明具有TAG合成酶活性；②上述5个NoDGAT对不同饱和度的脂酰CoA底物具有明显的偏好性差异；③将上述5个NoDGAT在微拟球藻中分别进行过表达时，可以促进微拟球藻的TAG含量提升，且不同NoDGAT转化株的TAG饱和度存在显著差异。本项目结合上述数据和NoDGAT时空表达信息，提出了微拟球藻TAG合成的机制模型，并建立了含有近百株NoDGAT突变体的藻株库。综上所述，本项目所有计划工作均已顺利完成，发表研究论文2篇（一区Top期刊， IF=10.812），申请专利3项。本项目阐明了微藻多DGAT的TAG合成机制，首次提出了油脂理性设计理念，并创制出多株具有潜在应用价值的工程株系，为基于DGAT的工业微藻油脂理性设计的深入开展打开了大门。