淫羊藿素和淫羊藿苷合成途径解析及微生物细胞工厂构建研究

Icaritin and icariin are the bioactive compounds of Chinese medicine Herba epimedii and the drug development of them has received much attention recently. Synthetic biology provide an alternative route for the production of natural products by using simple sugar as substrate, which will be a potential way to alleviate the problem of icaritin and icariin shortage caused by the insufficient supply of H. epimedii. In our previous work, we have already constructed yeast chassis strains for high-level production of naringenin and keampferol and characterized the key prenyltransferase involved in the biosynthesis of icariin. However, one methyltransferase and two glycosyltransferases which converting 8-prenylkeampferol into icaritin and icaritin into icariin respectively are necessary for the downstream pathway. In this study, we will firstly uncover the biosynthesis pathway of icaritin and icariin by data mining, screen and functional characterization of key methyltransferase and glycosyltransferase genes involved. Then based on the yeast chassis of keampferol, we will construct and optimize cell factories for de novo production of icaritin and icariin from glucose by fermentation. This study will not only lay a solid foundation for the environmental friendly manufacturing of icaritin and icariin in the future, but also provide platform for the studies of other complexed flavonoid compounds.

淫羊藿素和淫羊藿苷是中药淫羊藿的主要活性分子，近年来针对这些活性分子的药物开发受到普遍关注。合成生物学技术可以实现以单糖为原料高效合成特定天然产物，有望缓解由于药物开发可能引起的淫羊藿资源供应问题。前期研究中，我们构建了高效合成柚皮素和山奈酚的黄酮类化合物酵母底盘细胞，并完成了淫羊藿素合成途径中关键异戊烯基转移酶的鉴定。然而从8-异戊烯基山奈酚到淫羊藿素、从淫羊藿素再到淫羊藿苷还分别需要1个甲基转移酶和2个糖基转移酶参与。本研究中我们将通过数据挖掘，筛选并功能鉴定淫羊藿素和淫羊藿苷合成途径中所需要的关键甲基转移酶和糖基转移酶元件。在此基础上，于黄酮类化合物的酵母底盘细胞中重构淫羊藿素和淫羊藿苷的合成途径，并通过进一步优化细胞工厂与发酵工艺，实现以葡萄糖为原料从头合成淫羊藿素和淫羊藿苷。本研究将为淫羊藿素和淫羊藿苷的绿色制备奠定基础，并为其他复杂黄酮类化合物合成途径的解析和重构提供技术平台。

淫羊藿素和淫羊藿苷是中药淫羊藿的主要活性成分，具有抗肿瘤、预防骨质疏松、保肾等活性。尤其是淫羊藿素，作为晚期肝癌辅助阿可拉定的单一成分， 已于近期上市。然而，淫羊藿种植困难、化合物含量低导致当前植物提取模式极大的限制了淫羊藿素和淫羊藿苷的研究与应用。开展淫羊藿素和淫羊藿苷生物途径解析，探索其微生物异源合成具有重要意义。本项目旨在通过挖掘与鉴定淫羊藿素和淫羊藿苷合成途径中缺失的关键甲基转移酶与糖基转移酶元件，完成二者合成途径的解析，在此基础上，构建微生物细胞工厂，实现以微生物发酵从头合成淫羊藿素和淫羊藿苷。我们分别通过从大豆中鉴定了催化8P-KAE的C4’-OH甲基化的甲基转移酶GmOMT2，以及催化淫羊藿素两步糖基化合成淫羊藿苷的糖基转移酶UGT78D1和UGT73C6，首次获得了淫羊藿素和淫羊藿苷完整的生物合成路径。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。由于在弱酸性条件下GmOMT2的活性受到强烈抑制，导致其在8P-KAE酵母底盘细胞质中表达失去活性。通过将GmOMT2定位于弱碱性的线粒体中进行表达，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。通过将淫羊藿素细胞工厂与表达糖基转移酶的大肠杆菌细胞进行共培养，我们还首次实现了淫羊藿苷的从头合成。本研究微生物发酵法规模化制备淫羊藿素和淫羊藿苷奠定了基础，同时也为其它的异戊烯基修饰黄酮高效合成提供了指导。