青蒿二烯功能模块与酵母底盘的适配性

设计构建人工酿酒酵母细胞合成青蒿二烯的关键是使外源功能模块与底盘细胞适配，本文通过对外源功能模块中的载体、蛋白表达和启动子进行优化，以提高功能模块与底盘细胞的适配性. 使用着丝粒载体和附加型载体构建了2种青蒿二烯功能模块，在过表达甲羟戊酸（MEV）途径中关键基因（截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMGR及法尼基焦磷酸合酶基因ERG20）的2种酵母底盘中进行适配，得到适配性较好的人工合成细胞，其产量为11.2 mg/L；将青蒿二烯合酶基因（ADS）与ERG20进行融合构建融合蛋白功能模块，在选定底盘中适配性进一步提高，青蒿二烯的产量提升至17.5 mg/L；采用不同强度的启动子（TDH3p，TEF1p和PGK1p）对融合蛋白功能模块进行调控，最终得到功能模块与底盘间适配关系更好的人工合成细胞，其产量提升到71.8 mg/L.

Fitness of Amorphadiene Production Functional Modules and Yeast Chassis

The key to construct artificial yeast cells to produce amorphadiene is the fitness of heterologous functional modules and chassis. The fitness of functional modules and chassis were studied by optimization of vectors, protein expression and promoters in the functional modules. Two functional modules constructed with centromere vector and episomal vector were introduced into two engineered chassis overexpressing key genes(a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene tHMGR and farnesyl diphosphate synthase gene ERG20) in mevalonate(MEV) pathway, and the highest amorphadiene production was 11.2 mg/L in the artificial cell with better fitness. Amorphadiene synthase gene ADS and ERG20 were fused to construct fusion enzyme modules, and the fitness with the selected chassis were improved, resulted in a higher amorphadiene production of 17.5 mg/L. With promoter engineering(TDH3p, TEF1p, PGK1p) in the fusion enzyme modules, an artificial cell with a better fitness of functional module and chassis reached a 71.8 mg/L amorphadiene production.