人工细胞合成5α-羟化紫杉二烯的P450羟化酶特异性研究

Byproduct and low conversion effiency are the bottlenecks for the industrialized production of paclitaxel precursor produced by artificial cells. Based on the strategies of directed evolution and semi-rational design, the enzyme specificity of taxadiene 5α-hydroxylase will be improved to solve the above problems. Taking cytochrome P450 taxadiene 5α-hydroxylase as research object, this study attempt to do research from the following aspects: the construct of gene library of taxadiene 5α-hydroxylase by random mutation，getting the high-productivity strain by high- throughput screening method and mutation sites; the use of semi-rational design through molecular modeling and site-saturation mutagenesis to optimize taxadiene 5α-hydroxylase and look for mutation sites；rapid screening of the strains obtained through combinatorial optimization of the above mutation sites. Based on the results of these studies, the evolutionary mechanism of 5α-hydroxylase might be further explored. Our study will provide the foundation of highly efficient synthesis of taxol precursors by artificial cells, aiming at meeting the increasing demand in the world wide market.

人工细胞生产紫杉醇关键前体过程中存在副产物多，转化率低等问题，是制约利用人工细胞进行紫杉醇前体工业化大生产的瓶颈。申请者提出基于定向进化和半理性设计策略，对紫杉二烯5α羟化酶特异性进行改进，进而减少副产物、提高转化率以解决上述问题。本研究以催化紫杉醇关键前体紫杉二烯生成5α-羟基紫杉二烯的紫杉二烯5α-羟化酶为研究对象，尝试进行如下研究：采用多轮随机突变构建5α羟化酶的突变文库，高通量筛选5α-羟化紫杉二烯为主产物的高产菌株，获得突变位点；分子模拟结合定点饱和突变对5α羟化酶进行半理性设计，筛选突变酶；快速筛选上述突变位点随机组合获得的菌株并对突变体酶的表达条件进行优化；通过分子模拟解析突变酶催化产物组成发生改变的机制，揭示造成人工细胞合成5α-羟基紫杉二烯专一性差的原因，探索强化5α羟化酶催化特异性机制。该研究将为人工细胞高效合成紫杉醇前体、满足日益增长的需求提供技术基础。

人工细胞生产紫杉醇关键前体过程中存在副产物多，转化率低等问题，是制约利用人工细胞进行紫杉醇前体工业化大生产的瓶颈。本项目围绕催化紫杉醇关键前体紫杉二烯生成5α羟化紫杉二烯醇的紫杉二烯5α羟化酶体系展开研究。对涉及5α羟化紫杉二烯醇生物合成路径中各功能元件选择多个来源并优化，通过模块化组合设计获得了不同功能元件组合优化下的前体合成定量情况。通过半理性设计，采用N端穿膜工程引入不同来源和不同长度N端截短的紫杉二烯5α羟化酶与P450还原酶组合融合表达，发现紫杉二烯5α羟化酶的截短不超过42aa，可强化5α羟化酶催化特异性更利于5α羟化紫杉二烯醇的产生。该研究结果表明，利用组合设计策略研究模块间相互作用及其与底盘间适配性将为在微生物细胞中实现细胞色素P450酶系介导的催化反应过程提供重要参考，也为人工细胞高效合成紫杉醇前体、满足日益增长的需求提供技术基础。