一锅多酶体系催化C-H键胺化新途径的从头构建

Research on asymmetric C-H amination is always considered to be one the most challenging task. Single enzyme is still hardly to catalyze the C-H amination reaction efficiently and multi-enzyme-catalyzed cascade reaction for C-H amination will be the focused issue in the future biocatalysis research. In this project, we proposed a new multi-enzyme-catalyzed route for C-H amination reaction containing P450 monooxygenase, alcohol dehydrogenase and amine dehydrogenase, in which only ammonia was used as amino donor resulting easy product isolation and a clean reaction system. To solve the bottleneck problem of poor substrate adaptability and condition compatibility in the multi-enzyme catalytic system, we will focus on broadening the substrate profile and increasing the catalytic efficiency of P450LaMO by rational protein design and molecular modification since its poor substrate adaptability among the multiple enzymes; then we will enhance the enzyme’s tolerance by directed evolution with stress acclimation strategy to improve the compatibility of the combined reaction condition. On that basis, a green and efficiency process of C-H amination can be developed by optimizing this multi-enzyme-catalyzed reaction and constructing the multi-enzyme coexpressed artificial cell.

C-H键的不对称胺化始终是一项挑战性的工作，单个酶的作用很难实现C-H键的高效胺化反应，多酶组合催化C-H不对称胺化将成为未来研究的热点领域。本项目拟从头设计构建“P450单加氧酶+醇脱氢酶+酮胺化还原酶”的新型组合生物催化C-H胺化反应途径，直接使用无机铵为氨基供体，反应体系清洁环保，产物分离简便，应用前景广阔。针对组合催化体系中存在的多酶催化底物适配性差和反应条件不兼容的瓶颈问题，在前期考察各个酶底物特异性的基础上，对适配性较差的P450LaMO进行理性设计和分子改造，拓展其催化底物的范围，提高其催化效率；在考察组合反应条件下各个酶的耐受性及其失活原因的基础上，通过体外定向进化和逆境驯化筛选的策略获得耐受性更好的突变酶，以提高一锅组合反应的条件兼容性；在此基础上对上述多酶组合体系进行系统优化，最终构建一个高效、绿色的生物催化C-H胺化一锅反应新途径。

本项目以构建绿色高效的“P450单加氧酶+醇脱氢酶+胺化还原酶”多酶组合催化C-H胺化反应途径为目标。针对这一途径中多酶催化底物适配性差和反应条件不兼容的瓶颈问题，通过酶的构效关系解析和理性设计来改善了元件酶P450LaMO的底物适配性，通过元件筛选来提高多个酶在组合一锅反应体系中的兼容性，在此基础上通过对组合催化体系进行系统优化，实现这一组合体系中各个酶的协同运转。同时通过该项目的研究，为解决多酶组合催化体系普遍存在的“适配性、兼容性、协同性”关键问题形成有效的研究方法和可供借鉴的研究思路。本项目在Metabol. Eng.、ACS Catal.、ChemCatChem等期刊上发表论文15篇，授权发明专利4件，培养研究生 6名。