Complete biosynthesis of the potential medicine icaritin by engineered Saccharomyces cerevisiae and Escherichia coli  

创建工程酵母和大肠杆菌从头合成候选药物淫羊藿素

作　　者：Pingping Wang Chaojing Li Xiaodong Li Wenjun Huang Yan Wang Jiali Wang Yanjun Zhang Xiaoman Yang Xing Yan Ying Wang Zhihua Zhou 王平平;李超静;李晓东;黄文俊;王燕;王加莉;张燕君;杨晓满;严兴;王瑛;周志华(CAS-Key Laboratory of Synthetic Biology,CAS Center for Excellence in Molecular Plant Sciences,Institute of Plant Physiology and Ecology,Chinese Academy of Sciences,Shanghai 200032,China;University of Chinese Academy of Sciences,Beijing 100049,China;Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture,Wuhan Botanical Garden,Chinese Academy of Sciences,Wuhan 430074,China;School of Life Sciences,Henan University,Kaifeng 475001,China;Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement,Center of Economic Botany/Core Botanical Gardens,South China BotanicalGarden,Chinese Academy of Sciences,Guangzhou 510650,China)

机构地区：[1]CAS-Key Laboratory of Synthetic Biology,CAS Center for Excellence in Molecular Plant Sciences,Institute of Plant Physiology and Ecology,Chinese Academy of Sciences,Shanghai 200032,China [2]University of Chinese Academy of Sciences,Beijing 100049,China [3]Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture,Wuhan Botanical Garden,Chinese Academy of Sciences,Wuhan 430074,China [4]School of Life Sciences,Henan University,Kaifeng 475001,China [5]Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement,Center of Economic Botany/Core Botanical Gardens,South China BotanicalGarden,Chinese Academy of Sciences,Guangzhou 510650,China

出　　处：《Science Bulletin》2021年第18期1906-1916,M0004,共12页科学通报（英文版）

基　　金：supported by the National Key Research and Development Program of China (2018YFA0900700);the National Natural Science Foundation of China (31901021 and 31921006);the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27020206);the International Partnership Program of Chinese Academy of Sciences (153D31KYSB20170121);the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences (KFJ-BRP-009)。

摘　　要：Icaritin is a prenylflavonoid present in the Chinese herbal medicinal plant Epimedium spp. and is under investigation in a phase Ⅲ clinical trial for advanced hepatocellular carcinoma. Here, we report the biosynthesis of icaritin from glucose by engineered microbial strains. We initially designed an artificial icaritin biosynthetic pathway by identifying a novel prenyltransferase from the Berberidaceae-family species Epimedium sagittatum(EsPT2) that catalyzes the C8 prenylation of kaempferol to yield 8-prenlykaempferol and a novel methyltransferase GmOMT2 from soybean to transfer a methyl to C4’-OH of 8-prenlykaempferol to produce icaritin. We next introduced 11 heterologous genes and modified 12 native yeast genes to construct a yeast strain capable of producing 8-prenylkaempferol with high efficiency. GmOMT2 was sensitive to low pH and lost its activity when expressed in the yeast cytoplasm. By relocating GmOMT2 into mitochondria(higher pH than cytoplasm) of the 8-prenylkaempferol–producing yeast strain or co-culturing the 8-prenylkaempferol–producing yeast with an Escherichia coli strain expressing GmOMT2, we obtained icaritin yields of 7.2 and 19.7 mg/L, respectively. Beyond the characterizing two previously unknown plant enzymes and conducting the first biosynthesis of icaritin from glucose, we describe two strategies of overcoming the widespread issue of incompatible pH conditions encountered in basic and applied bioproduction research. Our findings will facilitate industrial-scale production of icaritin and other prenylflavonoids.淫羊藿素是中药淫羊藿的主要活性成分,作为晚期肝癌的候选药物已经进入Ⅲ期临床试验.本研究从淫羊藿中挖掘与鉴定了一个异戊烯基转移酶EsPT2,其高效催化山奈酚(KAE)的C8异戊烯基化合成8-异戊烯基山奈酚(8P-KAE);从大豆中鉴定了催化8P-KAE的C4’-OH甲基化的甲基转移酶GmOMT2,搭建了一条淫羊藿素的人工生物合成途径.通过引入11个外源基因以及改造12个酵母内源基因,构建了高产8P-KAE的酵母底盘.由于GmOMT2对低pH敏感,导致在酵母细胞质中表达失去活性.通过将GmOMT2定位于8P-KAE酵母底盘中具有更高pH的线粒体中,或将8P-KAE酵母底盘与表达GmOMT2大肠杆菌共培养,实现了淫羊藿素的合成,其产量分别为7.2和19.7 mg/L.该研究不仅鉴定了两个新的生物元件,首次实现了淫羊藿素的从头人工合成,为规模化制备淫羊藿素及其他异戊烯基化黄酮奠定了基础,同时也为解决合成生物学研究中普遍面临的生物元件pH不适配性问题提供了新策略.

关 键 词：淫羊藿素 Ⅲ期临床试验 晚期肝癌 合成生物学 异戊烯基转移酶 人工合成 甲基转移酶 从头合成 

分 类 号：R284[医药卫生—中药学]