基于酵母表达体系的葫芦二烯醇合成生物学研究

This project intends to investigate the synthesis of cucurbitadienol in yeast. Using the recombinant DNA technology, the key enzymes CS gene would adequately express in the yeast, and the regular pattern of high level synthesis of cucurbitadienol would be mastered. This study intends to solve the following problems: Screening of high yield squalene chassis strains of yeast, Squalene is a substrate of synthetic cucurbitadienol; Constructing of Saccharomyces cerevisiae deficient in lanosterol synthase gene ERG7, lanosterol synthase and CS competition the same substrate 2,3- oxidosqualene; Constructing of multigene co-expression vector; Comparing the differences of CS gene and CS gene expression among different plants, providing the basis for screening of CS and providing a new instance for enzyme protein structure-activity relationship research; Screening of chimeric promoter. Comprehensive coordination of various effect factors raise the yield of cucurbitadienol. These studies will not only greatly promote the Chinese medicine ingredient cucurbitadienol biosynthesis work, which will provide a scientific basis for the technical innovation of cucurbitadienol, but also for the study of yeast producing triterpene components of traditional Chinese medicine provides referential pattern and fresh experience.

本项目拟围绕酵母合成葫芦二烯醇存在的问题开展研究，通过DNA重组技术使合成关键酶CS基因在酵母中充分表达，掌握葫芦二烯醇高水平合成的规律，拟解决的问题有：筛选高产鲨烯的酵母底盘菌株，鲨烯是合成葫芦二烯醇底物；构建敲除ERG7基因的底盘菌，该基因编码的羊毛甾醇合酶(LSS)与CS竞争同一底物2,3-氧化角鲨烯；多基因共表达载体的构建；比较不同植物CS基因差异及表达差异，为CS的筛选提供依据，也为酶蛋白构效关系研究提供新实例；嵌合型启动子的筛选。综合协调各种作用因素大幅度提高葫芦二烯醇的产率。这些研究将极大地推进中药成分葫芦二烯醇生物合成工作，为其技术创新提供科学依据，同时也为利用酵母生产三萜类中药成分的研究提供可借鉴模式和新鲜经验。

本项目拟围绕酵母合成葫芦二烯醇存在的问题开展研究，通过DNA重组技术使合成关键酶CS基因在酵母中充分表达，掌握葫芦二烯醇高水平合成的规律，拟解决的问题有：筛选高产鲨烯的酵母底盘菌株，鲨烯是合成葫芦二烯醇底物；构建敲除ERG7基因的底盘菌，该基因编码的羊毛甾醇合酶(LSS)与CS竞争同一底物2,3-氧化角鲨烯；多基因共表达载体的构建；比较不同植物CS基因差异及表达差异，为CS的筛选提供依据，也为酶蛋白构效关系研究提供新实例；嵌合型启动子的筛选。综合协调各种作用因素大幅度提高葫芦二烯醇的产率。这些研究将极大地推进中药成分葫芦二烯醇生物合成工作，为其技术创新提供科学依据，同时也为利用酵母生产三萜类中药成分的研究提供可借鉴模式和新鲜经验。