在丰富萜类天然产物的化学多样性方面，生物合成相比传统的化学合成有诸多优势，但生物合成的成功应用还受限于随机性大、重现率低的缺陷，这主要是因为对萜类生源途径中相关酶催化机理的认识不够透彻。本项目拟将“机制-设计-合成”串联成一线来提高其成功率。以结构-功能相近的倍半萜环化酶ATAS和TEAS为研究对象，借助DFT/MM MD模拟，基于前期工作(Angew Chem和ACS Catal等)，继续深入阐明ATAS具有酶催化专一性(fidelity)而TEAS却呈现出催化杂泛性(promiscuity)的分子机制，揭示调控酶催化主副反应化学选择性的热动力学因素和关键残基，并通过酶突变实验加以验证。结合多尺度模拟及生物信息学方法，开展蛋白活性重设计，初步构建基于廉价工程菌的生物合成方案，力争获得若干个骨架特殊且有潜在药效的稠环倍半萜分子，为丰富倍半萜稠环骨架的化学多样性提供新策略。

Compared with chemical synthesis, the biosynthesis has been recognized as a more effective way to enhance the chemical diversity of terpenoid natural products. However, the successful application of synthetic biology is still limited by the big-randomicity and low-reproducibility, which highly depends on the exactly understanding of the biosynthetic machinery. Herein, "Mechanism-Design-Synthesis" would be integrated to enhance its success rate in this project. First, by DFT/MM MD simulations, we will elucidate the enzymatic catalysis fidelity/promiscuity of ATAS and TEAS which both belong to the Sesquiterpene Cyclase. Then computationally reveal the thermodynamical, kinetical and structural controllers on the chemical selectivity, and further proved by enzyme mutagenesis experiments. Finally, combined with multi-scale modeling and bioinformatics prediction, we will rationally redesign the enzymatic activity based on the enzyme catalysis mechanistic insights, and construct an efficient and low-cost engineering bacteria to biosynthesize the fused-ring sesquiterpene products with special skeleton and potential drug efficacy, as well as to expand the chemical space by trying to make novel fused-ring products.