王冬, 刘怡, 许骄阳, 王金鹤, 戴住波, 张学礼, 黄璐琦. 创建酿酒酵母细胞工厂高效生产人参皂苷前体达玛烯二醇IIJ. 药学学报, 2018,53(8): 1233-1241. doi: 10.16438/j.0513-4870.2018-0503
引用本文: 王冬, 刘怡, 许骄阳, 王金鹤, 戴住波, 张学礼, 黄璐琦. 创建酿酒酵母细胞工厂高效生产人参皂苷前体达玛烯二醇IIJ. 药学学报, 2018,53(8): 1233-1241. doi: 10.16438/j.0513-4870.2018-0503
WANG Dong, LIU Yi, XU Jiao-yang, WANG Jin-he, DAI Zhu-bo, ZHANG Xue-li, HUANG Lu-qi. Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-IIJ. Acta Pharmaceutica Sinica, 2018,53(8): 1233-1241. doi: 10.16438/j.0513-4870.2018-0503
Citation: WANG Dong, LIU Yi, XU Jiao-yang, WANG Jin-he, DAI Zhu-bo, ZHANG Xue-li, HUANG Lu-qi. Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-IIJ. Acta Pharmaceutica Sinica, 2018,53(8): 1233-1241. doi: 10.16438/j.0513-4870.2018-0503

创建酿酒酵母细胞工厂高效生产人参皂苷前体达玛烯二醇II

Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-II

  • 摘要: 人参皂苷是名贵药材西洋参和人参的主要活性成分,其中达玛烯二醇Ⅱ为人参皂苷类成分生物合成途径中的重要中间体。为构建高产人参皂苷前体达玛烯二醇Ⅱ的细胞工厂,本研究在优化过甲羟戊酸途径(MVA途径)工程菌的基础上,通过基因模块组合优化的方法获得能显著提高三萜前体的功能模块C30-M:分别由丹参法尼基焦磷酸合酶(SmFPS)和拟南芥鲨烯合酶(AtSQS2)组成,其鲨烯的含量能达到67.4 mg·g-1,约占细胞干重的6.74%;在进一步工作中,发现来源于拟南芥的2,3-环氧鲨烯合酶(AtSQE2)能提高下游中间体羊毛甾醇的产量到47.9 mg·g-1,为对照菌含量的22倍;然后,通过调控达玛烯二醇Ⅱ合酶基因的表达和采用反义RNA技术对麦角固醇合成途径中的ERG7基因进行调控及发酵工艺优化等策略,首次将三萜合成通量提高到10 g·L-1级别,创建出产量能达到15 g·L-1达玛烯二醇Ⅱ的高效酵母细胞工厂,为达玛烷型人参皂苷的产业化合成奠定了坚实的基础。

     

    Abstract: Dammarenediol-Ⅱ is an important precursor in the biosynthesis pathway of ginsenosides which are the main active components of Panax quinquefolius and Panax ginseng. For constructing a dammarenediol- Ⅱ-producing cell factory, the triterpenoid precursors of yeast are improved significantly by the modular pathway engineering strategy on the basis of an MVA optimized strain. The strain overexpressing Salvia miltiorrhiza SmFPS and Arabidopsis thaliana AtSQS2 could yield 67.4 mg·g−1 squalene, accounting for about 6.74% of cell dry weight. In our further work, an Arabidopsis thaliana 2,3-oxidosqualene synthase AtSQE2 was found to be able to increase the downstream lanosterol yield by 22-fold, reaching 47.9 mg·g−1. Then, regulating dammarenediol-Ⅱ synthase gene expression, using anti-sense RNA technology for regulation of ERG7 in the ergosterol pathway, and optimizing fermentation process were successively performed. Finally, the synthesis flux of triterpenes was increased to 10 g·L−1 for the first time, and we constructed an efficient cell factory that can produce 15 g·L−1 dammarenediol-Ⅱ, which lays a solid foundation of industrial synthesis of dammarane-type ginsenosides.

     

/

返回文章
返回