黄酮羟化酶底物识别机理与双加氧酶理性设计研究

Cytochrome P450 monooxygenase (P450) family is one of the most important protein family in synthesis of natural products. However, the identification, modification and optimization of P450 has become limiting factors in heterogeneous synthesis of natural products due to it’s cofactor-complexity and highly specific substrate-selectivity. In comparison, α-oxoglutarate-dependent dioxygenase (ODD) family exhibits more attractive shining points in catalyzing characters, such as robust cofactor reliability, no redox coenzyme consumption, and active expression in prokaryotes. Engineering ODD to substitute for P450 will help to solve problems in the heterogeneous synthesis of natural products. In this study, new ODD enzymes will be rational designed and engineered to catalyze apigenin-6-hydroxylation as a substitute for P450. Firstly, different flavonoid hydroxylases, both from P450 and ODD family will be revealed for their molecular mechanism of substrate adaptation. Secondly, anthocyanin synthase (ANS) will be chosen as template for new ODD apigenin-6-hydroxylase design, by using Rosetta platform. Finally, The new ODD F6H will be approached to directed evolution for better reaction activity through saturation mutagenesis, ProSAR modeling and DNA shuffling. This project will not only help to reveal substrates binding style and reaction mechanism of flavonoid hydroxylases, but also provide a new solution to solve P450 problems in natural products synthesis.

细胞色素P450酶是天然产物羟化反应的重要酶类。由于催化底物的高度专一性、反应体系的复杂性，P450的发掘鉴定与改造优化已经成为制约天然产物异源合成的重要科学瓶颈。α-酮戊二酸依赖的双加氧酶（ODD）具有与P450类似的催化功能，又具有易表达、反应体系简单等优点。若能以ODD酶替代P450酶将有助于突破天然产物异源合成的瓶颈问题。本研究拟设计ODD类新酶替代P450酶催化芹菜素6-羟基化反应：首先，解析P450和ODD类黄酮羟化酶底物结合模式，揭示黄酮类底物适配的分子机理；其次，以ODD类花青素合成酶ANS为模板，利用Rosetta平台辅助设计全新的ODD类芹菜素6-羟化酶（F6H）；最后，利用ProSAR建模定向进化提高ODD类F6H新酶活性。研究成果不仅有助于揭示黄酮羟化酶的底物识别机理，对黄酮6位羟基化催化机理研究具有重要意义，同时还将为解决P450酶催化的瓶颈问题开辟全新的思路。

细胞色素P450酶是天然产物羟化反应的重要酶类。由于催化底物的高度专一性、反应体系的复杂性，P450的发掘鉴定与改造优化已经成为制约天然产物异源合成的重要科学瓶颈。本研究首先解析了P450类黄酮羟化酶底物结合模式，揭示黄酮类底物适配的分子机理；其次，以EbF6H同家族基因但没有F6H催化功能的基因为模板，创建F6H新功能，解析了F6H功能起源机制；最后，利用ProSAR建模定向进化提高F6H新酶活性。研究成果不仅有助于揭示黄酮羟化酶的底物识别机理，对黄酮6位羟基化催化机理研究具有重要意义，同时还将为解决P450酶催化的瓶颈问题开辟全新的思路。通过本项目的支持，在Nature communications，Genome Biol. Evol，ACS Synthetic Biology等国际学术刊物上发表高质量研究论文4 篇，国内核心期刊发表论文2篇；申请专利2项；培养硕士/博士研究生5名。

内容获取失败，请点击重试