高效合成人参皂苷CK的酵母细胞工厂的研究

Compound K (CK) is one of the major ginsenoside-type metabolite (triterpene saponin) detected in blood or organs after oral administration of ginseng in mammals and thus regard as the main functional component in vivo; it is reported to possess bioactivities of anti-inflammation, hepatoprotection, anti-diabetes and anti-cancer. Recently synthetic biology provides available approaches to produce rare nature products with complex structures from glucose by microbial fermentation. In our previous study, several UDP-glycotransferases and two NADPH-cytochrome P450 reductases were identified from Panax ginseng and characterized, and thus a yeast cell factory to produce CK from glucose was also built. To improve the CK yield and titer of the yeast cell factory, it may be the key point to improve the pathways to supply precursors and energy for CK synthesis and the whole compatibility of cell factory. In this study, metabolic modifications would be carried out to strengthen the MVA pathway, the pathways to produce UDP-glucose and the expression level or activities of some key enzymes by repeating the process of design-modify-test. The newly built yeast cell factories would be characterized for its cell activities, gene expression level, enzyme activities and metabolite compositions, respectively. And thus the limited steps and the related key enzymes within the related pathways would be detected and tuned by modification of the expression level or activities of the key enzymes. The final objectives of the present project are to obtain an improved cell factory to produce CK with high efficiency and its optimized fermentation-conditions, and to provide improved yeast chasses and techniques to produce other terpene saponins from glucose.

人参皂苷CK是口服人参后在血液中检测到的主要四环三萜化合物，被认为是人参在体内代谢后被吸收到血液并发挥作用的主要活性分子之一。合成生物学为利用微生物发酵从单糖合成结构复杂的活性小分子化合物提供了新途径。在前期研究中我们克隆鉴定了一批人参皂苷糖基转移酶和细胞色素P450还原酶，构建了合成CK的酵母细胞工厂。优化合成CK所需要的前体和能量供给以及细胞工厂的整体适配性，是提高CK酵母细胞工厂产率的关键。本研究中，我们拟循环采用设计-改造-测试的方式，优化酵母细胞工厂的甲羟戊酸途径（MVA）和糖基供体UDP-glucose的供给，测试与分析细胞工厂在细胞、基因、蛋白和代谢物水平上的表型，确定影响细胞工厂适配性的关键因素，通过启动子置换调节相关限速酶的表达水平，最终获得高产CK的酵母细胞工厂及其最佳发酵工艺，并为从头合成其他萜类天然产物提供高效、通用的底盘细胞。

稀有人参皂苷CK具有抗癌、保肝、抗炎症和治疗糖尿病等多种活性，然而其在人参属植物中含量极低，无法检测，只能通过从人参属植物中提取的其它人参皂苷转化而来。这种传统的制备方式周期长、成本高且难以规模化，极大地限制了CK的新药研发和应用。本项目在前期解析稀有人参皂苷CK合成途径解析和初代细胞工厂的基础上，优化合成CK所需要的前体和能量供给以及细胞工厂的整体适配性，以进一步提高CK合成效率。采用设计-改造-测试的循环改造方式，我们首先通过模块化的方式对前体MVA途径进行整体优化和细胞色素P450的表达优化等策略构建了高效合成PPD底盘细胞，其摇瓶发酵产量达到570 mg/L。随后基于该底盘我们进行了第三代CK细胞工厂（ZWCK3）的构建，通过优化UGTPg1表达水平CK产量比第二代细胞工厂提高了1.1倍达到了101.9 mg/L。基于对第三代CK细胞工厂的测试与分析，我们随后对其UDP-glucose的供给模块进行了系统的优化，获得了第四代CK细胞工厂（ZWCK4），CK产量进一步提高为145.5 mg/L。随后以第四代CK细胞工厂为发酵菌株，我们开展了5L，10L和50L发酵测试和工艺优化，确定了CK细胞工厂的最佳发酵工艺，最终CK产量达到2.8 g/L。该产量不仅是目前利用合成生物学技术合成稀有人参皂苷最高产量，而且与现有工艺相比也初步具备了成本优势，达到了经济可行水平。本项目的开展为稀有人参皂苷CK提供了一条极具潜力的绿色、持续稳定的生产工艺，同时所构建的系列酿酒酵母底盘和获得的基础数据，也为其他萜类化合物和具有糖基修饰的天然化合物的人工合成提供了指导。