Abstract: In recent years,producing terpenoids in microbe by synthetic biology methods has become more and more popular.Inducible promoters and constructive promoters have both advantages and disadvantages. The choice of promoters depends on the specific systems and target compounds.In this research,regulations of heterologous modules by inducible and constructive promoters were studied respectively for the taxadiene synthesis in Saccharomyces cerevisiae.A yeast chassis was firstly constructed after expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene(thmgr),endogenous farnesyl diphosphate synthase gene(erg20)under the regulation of tdh3 promoter.After inserting the heterologous module in which ts was regulated by inducible promoter into the genome of chassis,an engineered strain which produced 5.2 mg·L^-1 taxadiene was obtained.When the promoter was replaced by constructive one-tdh3p,the yield increased to 11.5 mg·L^-1.That indicated the constructive promoter may be more suitable for the taxadiene synthesis system in yeast.After a simple fermentation optimization in shaking flask,the yield increased by 70% to reach 19.5 mg·L^-1.Strains regulated by constructive promoters can use glucose as carbon source which was suitable for large scale fermentation in future.

Key words: taxadiene, yeast chassis, functional module, inducible promoter, constructive promoter

摘要： 近年来利用合成生物学手段生产萜类化合物逐渐成为一种趋势。诱导型和组成型启动子调控萜类的生产各有利弊,而启动子类型的选择需要根据特定体系和产物来进行具体分析。本工作针对紫杉醇关键前体紫杉二烯的合成,分别研究了酵母细胞中诱导型启动子和组成型启动子的调控。首先通过过表达酵母内源截短的羟甲戊二酰辅酶A基因(thmgr)和法尼基焦磷酸合酶基因(erg20),实现了对酵母内源模块的调控。随后向改造后的底盘细胞中整合入诱导性启动子调控的外源紫杉二烯合成模块,得到了紫杉二烯产量为5.2 mg·L^-1的人工酵母。而换用组成型启动子tdh3p调控时,产量达到11.5 mg·L^-1,说明对于这个体系组成型启动子更为适合。通过简单的发酵条件优化,该菌株产量提升了70%,达到19.5 mg·L^-1。组成型启动子调控的菌株可以利用葡萄糖作为碳源,因而更利于后续大规模发酵。

关键词: 紫杉二烯, 酵母底盘, 功能模块, 诱导型启动子, 组成型启动子