酿酒酵母高效合成甘草次酸的途径设计与优化

Glycyrrhetinic acid, the main modified ingredient of traditional Chinese medicinal plant licorice, is kind of triterpenoids. Because of its extensive pharmaceutical activities and special structure, glycyrrhetinic acid has been widely used in medicine, cosmetics, food and other fields. Presently, glycyrrhetinic acid is primarily produced by hydrolyzing glycyrrhizin extracted from licorice, which is strongly depended on licorice resource, resulting in severe ecological destroy. However, this producing method is gradually limited by increased scarce licorice resource. Previously, we constructed and obtained an engineered Saccharomyces cerevisiae with the capacity of producing triterpenoid skeleton, β-amyrin. Therefore, this project intends to explore the synthesis of glycyrrhetinic acid in Saccharomyces cerevisiae. To do this, the key enzymes including cytochrome P450 monooxygenase and dehydrogenase for glycyrrhetinic acid biosynthesis will be explored using various ways. After researches of catalytic characteristics of target function enzymes, the optimal methods of enzymes will be studied. For efficient expression of cytochrome P450 monooxygenase and dehydrogenase, they will be artificially designed and constructed with a combination of modular method, and finally create glycyrrhetinic acid biosynthetic pathway. Through assembling glycyrrhetinic acid biosynthetic pathway into engineered Saccharomyces cerevisiae which can supply the precursor of glycyrrhetinic acid biosynthesis, the glycyrrhetinic acid biosynthesis in Saccharomyces cerevisiae expects to be achieved. The adaptive relations among various synthesis module and the yeast endogenous pathway will be focused so as to establish a method for balance and regulate glycyrrhetinic acid pathways and finally improve the biosynthesis efficiency of glycyrrhetinic acid in Saccharomyces cerevisiae. This study expect to provide a theoretical and technical basis for the use of engineered Saccharomyces cerevisiae producing secondary metabolites such as terpenes.

甘草次酸是中国传统药用植物甘草的主要改性成分，属于三萜类化合物，由于其广谱的药物活性和特殊的中间体结构被广泛地应用于医药、化妆品和食品等领域。目前以甘草酸水解生产甘草次酸的方法对甘草资源的依赖性很强、生态破坏严重，且日益出现的甘草资源稀缺已大大限制了其可持续发展。在前期构建的酿酒酵母工程菌合成三萜骨架β-香树脂醇的基础上，本项目拟通过发掘甘草次酸生物合成的关键酶细胞色素P450单氧化酶和脱氢酶，研究目标酶的催化特性并进行酶工程优化，开展细胞色素P450单氧化酶和脱氢酶的人工模块化设计与组合表达，创建甘草次酸的酵母细胞合成途径，通过与前期构建的三萜骨架合成途径的适配性组装，实现甘草次酸在酿酒酵母中的人工合成，并通过研究合成模块与酵母内源竞争途径之间的互适应关系，探索平衡甘草次酸代谢途径的优化方法，提高甘草次酸的合成效率。研究结果也为利用酿酒酵母合成其它萜类化合物提供依据和思路。

甘草次酸是中国传统药用植物甘草的主要改性成分，属于三萜类化合物，由于其广谱的药物活性和特殊的中间体结构被广泛地应用于医药、化妆品和食品等领域。目前以甘草酸水解生产甘草次酸的方法对甘草资源的依赖性很强、生态破坏严重，且日益出现的甘草资源稀缺已大大限制了其可持续发展。本研究为了实现植物天然产物甘草酸的发酵法生产，利用合成生物学技术和代谢工程手段构建甘草次酸的酵母合成体系。研究获得了生物合成甘草次酸的关键酶P450氧化酶和相应的P450还原酶；利用模块化设计策略，完成了甘草次酸及其11-氧-β-香树脂醇合成途径的构建，实现了它们的人工合成；优选获得了生物合成甘草次酸的底盘宿主和途径表达方式；发现了甘草次酸生物合成的关键限速步骤，并建立了半理性改造该限速细胞色素单加氧酶CYP72A154的方法，获得了有益突变，使甘草次酸的合成增加了1.60倍；结合调节细胞氧化还原状态和平衡NADPH还原力，有效提高了11-氧-β-香树脂醇和甘草次酸的合成，最后在乙醇补料发酵模式下，甘草次酸的合成水平达到18.9 mg/L。研究结果为甘草次酸的合成提供了一种新方法，也为利用酿酒酵母合成其它萜类化合物提供依据和思路。