创建酿酒酵母细胞工厂发酵生产羽扇豆醇

Construction of cell factories for production of lupeol in Saccharomyces cerevisiae

羽扇豆醇、桦木酸等羽扇豆型三萜化合物具有抗HIV、抗肿瘤等多种生物学活性,其中羽扇豆醇为这类化合物的基本前体。为了实现羽扇豆烷型三萜的异源发酵法生产,研究首先运用高通量同源重组法在酿酒酵母中进行萜类甲羟戊酸(MVA)途径的一步法调控,以提高三萜通用前体鲨烯的供给;在进一步工作中,拟南芥来源的羽扇豆醇合成酶基因(At LUP)被整入三萜底盘菌株中实现羽扇豆醇酵母人工细胞工厂的创建。结果表明该实验能一次完成MVA途径的7个基因的整合,组装总长度达到20kb,同时多倍化MVA途径能显著提高鲨烯产量约500倍,达到354.00 mg·L^(-1);At LUP基因在染色体上整合后获得的工程菌NK2-LUP在摇瓶中发酵能生产8.23 mg·L^(-1)羽扇豆醇。该研究可为在酵母中实施大规模生物合成途径组装提供技术支持,同时为进一步获高产羽扇豆烷型三萜的人工酵母细胞提供了重要基础。

Lupane-type triterpenoids,such as betulinic acid,are derived from lupeol and have excellent properties in anti-HIV,anti-cancer activities and so on. For realizing heterogenous production of lupane-type triterpenoids,our research firstly integrated all the seven genes in the MVA pathway in Saccharomyces cerevisiae to increase the supply of squalene（ triterpenoids universal precursor） in a single step using the DNA assembler method. Next,cell factories for production of lupeol was constructed by integrating Arabidopsis thaliana lupeol synthetic gene（ At LUP） into chromosome of triterpenoid chassis strain. Results showed that the MVA pathway,about20 kb nucleotide length,could be assembled in one-pot process and the doubled MVA pathway could significantly improve squalene by500-fold,reaching 354. 00 mg·L^（-1）. NK2-LUP was obtained by introducing At LUP gene on chromosome,and could produce 8. 23 mg·L^（-1）lupeol. This study supports the possibility of large-scale biosynthetic pathway assembly in S. cerevisiae and lays the foundation of obtaining cell factories for production of lupan-type triterpenoids at the same time.