Wang Pingping, Li Chaojing, Li Xiaodong, Huang Wenjun, Wang Yan, Wang Jiali, Zhang Yanjun, Yang Xiaoman, Yan Xing, Wang Ying, Zhou Zhihua
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.
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.
Sci Bull (Beijing). 2021 Sep 30;66(18):1906-1916. doi: 10.1016/j.scib.2021.03.002. Epub 2021 Mar 7.
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.
淫羊藿素是一种存在于中国草药植物淫羊藿属中的异戊烯基黄酮，目前正在进行一项针对晚期肝细胞癌的III期临床试验。在此，我们报道了通过工程微生物菌株从葡萄糖生物合成淫羊藿素的过程。我们最初通过从小檗科植物箭叶淫羊藿（EsPT2）中鉴定出一种新型异戊烯基转移酶来设计一条人工淫羊藿素生物合成途径，该酶催化山奈酚的C8异戊烯基化反应生成8 - 异戊烯基山奈酚，以及从大豆中鉴定出一种新型甲基转移酶GmOMT2，将甲基转移到8 - 异戊烯基山奈酚的C4'-OH上以产生淫羊藿素。接下来，我们引入了11个异源基因并修饰了12个天然酵母基因，构建了一种能够高效生产8 - 异戊烯基山奈酚的酵母菌株。GmOMT2对低pH敏感，在酵母细胞质中表达时会失去活性。通过将GmOMT2重新定位到产生8 - 异戊烯基山奈酚的酵母菌株的线粒体中（线粒体pH高于细胞质），或者将产生8 - 异戊烯基山奈酚的酵母与表达GmOMT2的大肠杆菌菌株共培养，我们分别获得了7.2和19.7 mg/L的淫羊藿素产量。除了表征两种先前未知的植物酶并首次实现从葡萄糖生物合成淫羊藿素外，我们还描述了两种克服基础和应用生物生产研究中普遍存在的pH条件不兼容问题的策略。我们的研究结果将有助于淫羊藿素和其他异戊烯基黄酮的工业规模生产。
