[目的] 摩尔酸作为齐墩果烷型三萜化合物具有抗HIV、抗炎等多种生物学活性，其前体物质是计曼尼醇，本研究基于合成生物学策略构建酿酒酵母细胞工厂高效合成摩尔酸。[方法] 运用CRISPR/Cas9技术，首先分别整合不同来源的氧化鲨烯环化酶（OSCs），筛选高产计曼尼醇底盘细胞；进一步异源表达长春花来源的细胞色素P450氧化酶（CYP716AL1）和麻风树来源的细胞色素P450还原酶（JcCPR），构建摩尔酸生物合成途径；并通过CYP716AL1和不同来源的CPR适配研究以及过表达甲羟戊酸（MVA）代谢途径中关键酶的方式提高摩尔酸的产量。[结果] 整合苹果来源的氧化鲨烯环化酶MdOSC获得的重组菌株计曼尼醇产量最高，达68.3 mg/L；以此为底盘细胞进一步整合CYP716AL1和JcCPR实现了摩尔酸的生物合成，产量为15.0 mg/L；共表达CYP716AL1和拟南芥来源的CPR获得的重组菌株摩尔酸产量最高，达到24.3 mg/L；最后过表达MVA代谢途径中的关键酶法呢基焦磷酸合酶（ERG20）和鲨烯环氧酶（ERG1），获得的重组菌株摩尔酸产量高达34.1 mg/L。[结论] 本研究实现了摩尔酸的高效生物合成，为构建高产齐墩果烷型三萜酿酒酵母细胞工厂提供了理论和技术依据。
[Objective] Morolic acid is derived from germanicol, has excellent anti-HIV and anti-inflammatory activities properties as oleanane-type triterpenoid, thus their potential applications in the pharmaceutical industry. In this study, synthetic biology was used to construct Saccharomyces cerevisiae cell factories for efficient biosynthesis of morolic acid. [Methods] Firstly, using CRISPR/Cas9 technology, three oxidosqualene cyclases (OSCs), namely MdOSC, PbOSC and RcOSC were integrated into S. cerevisiae to screen for high yield germanicol chassis cells. Then, to construct the engineered strain capable of morolic acid production, the cytochrome P450 oxidase (CYP716AL1) derived from Catharanthus roseus and the cytochrome P450 reductase derived from Jatropha curcas (JcCPR) were further integrated into S. cerevisiae. Moreover, to improve morolic acid biosynthesis, the CYP716AL1 was coexpressed with CPRs (AtCPR, LjCPR, GuCPR and MtCPR) derived from different plants. Finally, the mevalonate (MVA) pathway was modified by overexpressing the key pathway enzymes to improve morolic acid production. [Results] The engineered strain S201 obtained by integrating oxidosqualene cyclase MdOSC from Malus domestica yielded the highest amount of germanicol of 68.3 mg/L. The engineered strain S401 obtained by integrating CYP716AL1 and JcCPR in S201 achieved biosynthesis of morolic acid, and the yield reached 15.0 mg/L. Furthermore, the CYP716AL1 was coexpressed with AtCPR from Arabidopsis thaliana in S201, resulting in engineered strain S402, which yielded the highest amount of morolic acid of 24.3 mg/L. Finally, overexpression of key enzymes in the metabolic pathway of MVA in engineered strain S402, FPP synthase (ERG20) and squalene epoxidase (ERG1), resulted in a morolic acid yield of 34.1 mg/L (S6). [Conclusion] In this study, morolic acid cell factories were successfully constructed, which provides a theoretical and technical basis for the construction of high-yield oleanane-type triterpenoids cell factories.