酵母生产五环三萜类化合物的胞外转运与合成强化

Pentacyclic triterpenoids have beneficial and extensive pharmaceutical activities such as anti-inflammatory, anti-bacteria and anti-fungi, liver protection and immunity enhancement. The extraction of pentacyclic triterpenoids from either natural plants or chemical synthesis can not meet the big market demand. Thus, microbial synthesis of pentacyclic triterpenoids has become a prime target for metabolic engineering. However, the low yield and low productivity resulting from the inefficient transportation outside the cell in biosynthesis process has greatly limited its industrial applications. Therefore, in this project we use β-amyrin, an economically important pharmaceutical pentacyclic triterpenoid serving as the key skeleton in many pentacyclic triterpenoids biosynthesis, as the model product and the previously engineered Saccharomyces cerevisiae as the chassis, in order to explore key transporters of β-amyrin in yeast and to study their functions as well as the applications using synthetic biology. The efficient extracellular transportation system of β-amyrin in engineered yeast will be established by exploring big data and expressing transportor proteins related to pentacyclic triterpenoids, and rationally designed and optimized combined with the adaptive assembly in biosynthesis pathway following structure analysis and modifications. Furthermore, the investigation will focus on the adaptive relations between the biosynthesis and extracellular transportation of β-amyrin so as to enhance the production in yeast cells. Aiming to clarify the transport mechanisms of pentacyclic triterpenoids, this study will achieve the extracellular transportation of β-amyrin in yeast and establish the most optimal producing model for the combination of β-amyrin biosynthesis within the cell and its transportation from cell to the outside. The study will not only be useful to break the bottleneck in further improvement of yield of β-amyrin, but also provide new methods and platfroms for the transmembrane transportation of other pentacyclic triterpenoids.

五环三萜类化合物具有抗炎、抗菌、护肝、提高免疫力等良好的药物活性，从天然原料中提取或化学合成存在含量低、副产物多、环境污染等问题，而利用微生物生产也因在胞内合成，很难转运到胞外而导致产量和生产效率低，严重限制了其工业化应用。为解决这一问题，本项目拟选取在五环三萜类化合物合成中具有核心骨架作用的β-香树脂醇为模式产物，以前期构建的生产β-香树脂醇的酵母为底盘，运用合成生物学方法开展β-香树脂醇转运蛋白发掘与功能应用的研究。通过大数据挖掘和表征与五环三萜类化合物相关转运蛋白，并进行结构分析与改造，构建酵母细胞β-香树脂醇胞外转运系统；通过人工设计优化并与合成途径适配性组装提高转运效率；研究合成与转运的关系以强化产物合成。本项目将实现β-香树脂醇在酵母中的胞外转运，阐明五环三萜类化合物的转运机制，建立合成与转运的最优生产模式，打破产量再提高的瓶颈，为其他五环三萜类化合物的跨膜转运提供思路和方法。

五环三萜类化合物具有抗炎、抗菌、护肝、提高免疫力等良好的药物活性，从天然原料中提取或化学合成存在含量低、副产物多、环境污染等问题，而利用微生物生产也因在胞内合成，很难转运到胞外而导致产量和生产效率低，严重限制了其工业化应用。为解决这一问题，本项目以前期构建的可以合成β-香树脂醇、α-香树脂醇、甘草次酸等五环三萜类化合物的酿酒酵母为底盘，运用合成生物学与生物信息学方法开展五环三萜类化合物转运蛋白发掘与功能应用的研究。利用大数据挖掘、转录组分析等方法获得与五环三萜类化合物相关转运蛋白，并进行功能表征；通过基于机器学习的转运蛋白预测方法的建立挖掘和表征针对不同底物的转运蛋白；建立β-香树脂醇在酿酒酵母中的外排系统，并通过优化外排系统与胞内合成途径，提高了β-香树脂醇的产量。本项目从解决因产物胞内积累造成的生产效率低、后处理困难等问题出发，研究了五环三萜化合物在酿酒酵母中的转运过程，建立合成与转运的最优生产模式，打破产量再提高的瓶颈。以上研究还为其他天然产物的胞外转运提供了新思路和新方法。