7-脱氢胆甾醇合成功能模块与底盘细胞的适配性

利用合成生物技术生产7-脱氢胆甾醇的挑战性在于获得合成功能模块与底盘细胞的适配关系。从更换不同调控强度的启动子和不同改造的酵母底盘两方面,对二者的适配性进行研究,以增加7-脱氢胆甾醇产量:过表达酵母固醇合成途径中的内源基因tHMGR和ERG1,敲除非必需基因ERG6和ERG5以抑制酵母固醇向麦角固醇的转化,得到改造后的酵母底盘SyBE_000956;利用由强到弱依次为TDH3p、PGK1p和TDH1p的启动子,引入人源C-24还原酶基因DHCR24,构建3种强度的外源功能模块,并分别导入3种底盘中,得到9种人工合成细胞。结果表明,TDH3p调控的功能模块与底盘细胞SyBE_000956具备较好的适配性,实现7-脱氢胆甾醇产量的提高。为后续的适配性研究提供了理性设计的依据。

Match of functional module with chassis in 7-dehydrocholesterol synthesis

The key challenge to generate engineered cells by synthetic biology for producing 7-dehydrocholesterol (7-DHC) in a high titer is the match between functional module and chassis. Our study focused on solving this problem by combining different promoters and yeast chassis to increase 7-DHC production. To optimize the chassis in order to accumulate zymosterol, the substrate for 7-DHC synthesis, we overexpressed truncated HMG-CoA reductase (tHmg1p) and squalene epoxidase (Erg1p), both are key genes of yeast endogenous zymosterol biosynthetic pathway. In addition, we knocked out C-24 methyl transferase (Erg6p) and C-22 dehydrogenase (Erg5p) to inhibit the conversion of zymosterol to ergosterol. By introducing heterologous C-24 reductase under three promoters with different strengths, namely TDH3p, PGK1p and TDH1p, we constructed functional modules of diverse activities. Nine engineeredcells were generated based on the combination of these three modules and three chassis. The result shows that the engineered cell composed of functional module regulated by TDH3p and chassis SyBE_000956 had the highest 7-DHC production, indicating a better match than others. This study provides evidences for importance of match and empirical support for rational design of subsequent researches.