产瓦伦西亚烯酿酒酵母细胞对CEN/ARS元件的应答响应研究

Saccharomyces cerevisiae is considered as the ideal host to produce nature terpenoids. Studies about yeast cell factory were almost based on plasmids. Variability of plasmid units brings convenience, but also accompanied with slow growth and metabolic burden. In our previous study, final cell density and valencene production were both increased when the engineered Saccharomyces cerevisiae cells harboring the CEN/ARS type plasmid, which is not exactly the same with traditionally “plasmid burden”. In this study, plasmids containing different sequence CEN/ARS would be transformed into Saccharomyces cerevisiae. Cell growth and valencene production will be recorded. The key element and best sequence would be identified. The key signal molecules would also be determined through transcriptome and metabolic analysis, which help elucidate response mechanism of Saccharomyces cerevisiae to CEN/ARS plasmid. To validate the responding mechanism, genome edited Saccharomyces cerevisiae mutants will also be constructed. Achievements in this project will expand the response theory of plasmid-yeast cell, and also help design efficient terpenoids yeast cell factory.

酿酒酵母被认为是合成天然萜类化合物的理想宿主细胞，围绕酵母细胞工厂的诸项工作均以质粒作为载体展开。质粒元件的灵活可变在给研究带来便利的同时，也给酵母细胞带来诸如生长缓慢、代谢流改变等潜在问题。前期研究中，申请人发现CEN/ARS型复制起始位点可刺激酿酒酵母细胞生长和瓦伦西亚烯积累，该现象与常规理解的质粒“负担”不完全一致。本项目将不同序列来源的CEN/ARS型质粒导入酿酒酵母细胞，考察细胞生长和瓦伦西亚烯积累量的变化，鉴定关键作用元件，确定适宜瓦伦西亚烯积累的最佳CEN/ARS序列；利用转录组学和代谢组学结合分析，从分子和代谢水平定位关键信号分子，理论上解析酿酒酵母细胞对质粒CEN/ARS元件的细胞应答响应机制；重构酿酒酵母工程菌，实验验证细胞对质粒CEN/ARS元件的响应机制。该研究结果将填补酿酒酵母细胞-质粒应答响应理论，为其他萜类化合物的高效酵母细胞工厂构建提供借鉴。

本项目以CEN/ARS型质粒“刺激”酵母细胞生长及瓦伦西亚烯积累为背景研究酿酒酵母细胞-质粒应答响应理论机制。通过生物信息学大数据分析、分子克隆、基因组编辑、天然启动子库筛选与杂交等技术手段、气相、酶活、荧光、Real-time PCR 等检测方法，从分子表征、生化检测和代谢物分析探究了酵母细胞生长与产物代谢对外源质粒的响应机制，鉴定了关键作用元件，分析了关键序列，揭示了外源质粒CEN/ARS元件在重塑酵母三维基因组、影响基因表达调控方面的生物物理机制。基于理论基础研究成果，指导重构了酿酒酵母工程菌，获得了高产瓦伦西亚烯的酵母菌，产量超过10 g/L，达到产业化水平。项目研究期间，申报人以通讯作者身份发表研究论文13篇，其中SCI论文9篇和中文核心论文4篇。其中1篇SCI影响因子大于10分，5篇为中科院分区1区论文。其中发表在Green Chemistry的文章受到科技部中国生物技术发展中心新闻报道。申请发明专利10项，其中1项已授权。登记软件著作权1项。项目负责人入选2019年广东省特支计划科技创新青年拔尖人才项目。培养博士生2名，硕士生3名。