Conflict between cell growth and product accumulation is frequently encountered in the biosynthesis of secondary metabolites. To address the growth-production conflict in yeast strains harboring the isoprene synthetic pathway in the mitochondria, the dynamic control of isoprene biosynthesis was explored. A dual temperature regulation system was developed through engineering and expression regulation of the transcriptional activator Gal4p. A cold-sensitive mutant, Gal4ep19, was created by directed evolution of Gal4p based on an internally developed growth-based high-throughput screening method and expressed under the heat-shock promoter P_SSA4 to control the expression of P_GAL-driven pathway genes in the mitochondria. Compared to the control strain with constitutively expressed wild-type Gal4p, the dual temperature regulation strategy led to 34.5% and 72% improvements in cell growth and isoprene production, respectively. This study reports the creation of the first cold-sensitive variants of Gal4p by directed evolution and provides a dual temperature control system for yeast engineering that may also be conducive to the biosynthesis of other high-value natural products.

在次生代谢物的生物合成中经常遇到细胞生长和产物积累之间的冲突。为了解决线粒体中含有异戊二烯合成途径的酵母菌株的生长-生产冲突，探索了异戊二烯生物合成的动态控制。通过转录激活因子 Gal4p 的工程化和表达调控开发了双温度调控系统。冷敏感突变体 Gal4ep19 是基于内部开发的基于生长的高通量筛选方法通过 Gal4p 定向进化产生的，并在热休克启动子 P _SSA4下表达以控制 P _GAL的表达线粒体中的驱动通路基因。与具有组成型表达的野生型 Gal4p 的对照菌株相比，双重温度调节策略分别使细胞生长和异戊二烯产量提高了 34.5% 和 72%。这项研究报告了通过定向进化产生的第一个冷敏感型 Gal4p 变体，并为酵母工程提供了双温度控制系统，这也可能有助于其他高价值天然产物的生物合成。