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

作　　者：王冬[1,2] 刘怡[1,2] 许骄阳[1,2] 王金鹤 戴住波 张学礼[1,2] 黄璐琦[3] WANG Dong;LIU Yi;XU Jiao-yang;WANG Jin-he;DAI Zhu-bo;ZHANG Xue-li;HUANG Lu-qi(Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;Key Laboratory of Systems Microbial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国科学院天津工业生物技术研究所,天津300308 [2]中国科学院系统微生物工程重点实验室,天津300308 [3]中国中医科学院中药资源中心,北京100700

出　　处：《药学学报》2018年第8期1233-1241,共9页Acta Pharmaceutica Sinica

基　　金：中国科学院青年创新促进会项目(2015138)

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(Sm FPS)和拟南芥鲨烯合酶(At SQS2)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(At SQE2)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。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 Sm FPS and Arabidopsis thaliana At SQS2 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 At SQE2 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.

关 键 词：人参皂苷 达玛烯二醇Ⅱ 酿酒酵母 合成生物学 微生物细胞工厂 

分 类 号：R931[医药卫生—生药学]