基于酶的定向挖掘及理性设计生物合成鞣花酸及其衍生物

Ellagic acid and its derivatives are important natural products that have high bio-safety and attractive activities such as anti-cancer, whitening and uric acid-lowering. They are widely used in medicine, health care and cosmetics industries. Biosynthesis of those products has not yet been achieved due to their natural synthetic pathways have not been resolved so far. Based on our previous work, we intend to design artificial biosynthetic pathways for the synthesis of ellagic acid and its important derivative 3,3'-dimethyl ellagic acid via combining “top-down” combinatorial and “bottom-up” reverse synthetic strategies. However, the key enzymes 4-O-methyltransferase and peroxidase are suffered low catalytic efficiency and poor specificity. The project intends to obtain high efficient peroxidase by enzyme directed excavation. Then, the catalytic mechanism and specificity of 4-O-methyltransferase and peroxidase will be revealed by structural analysis, homology modeling and molecular docking. Rational protein engineering will be used to obtain more efficient and specific enzyme mutants. based on this, the platform strains for efficient biosynthesis of ellagic acid and its derivatives will be established by construction of de novo pathways and introduction of the cofactor regeneration systems. This work will lay a theoretical and technical basis for large-scale production of ellagic acid and its derivatives, and also provides new ideas and methods for the biosynthesis of natural products with unknown synthetic pathways.

天然产物鞣花酸及其衍生物具有抗癌、美白和降尿酸等重要活性，安全性高，被广泛应用于药品、保健品及化妆品行业。有研究者提出了其可能的天然合成途径，但至今未被证实，生物合成尚未实现。本项目以课题组前期工作为基础，拟结合“自上而下”的组合生物合成及“自下而上”的逆合成策略设计构建鞣花酸及其重要衍生物3,3’-二甲氧基鞣花酸的人工合成途径，但关键酶4-O-甲基转移酶和过氧化物酶存在催化效率低、专一性差等问题。项目拟通过酶定向挖掘策略获得目标过氧化物酶，利用结构分析、同源建模等手段解析4-O-甲基转移酶和过氧化物酶的催化机理和专一性分子机制，理性设计得到高效、专一的酶突变体；构建鞣花酸及3,3’-二甲氧基鞣花酸的从头合成途径，引入辅因子再生系统提高途径生产效率，获得可生产鞣花酸及其衍生物的高效平台菌株，为其大规模生产应用奠定理论和技术基础，也为代谢途径未知的天然产物的生物合成提供新思路和新方法。

天然产物鞣花酸及其衍生物具有抗癌、美白和降尿酸等重要生物活性，安全性高，被广泛应用于药品、保健品及化妆品行业。由于其天然生物合成途径未被解析，微生物合成鞣花酸及其衍生物尚未实现。本项目设计构建了鞣花酸及其重要衍生物3,3’-二甲氧基鞣花酸的人工合成途径。通过筛选途径关键酶、增加前体供应、敲除竞争性途径、PTS系统改造等代谢工程策略，实现了鞣花酸前体物质没食子酸的高产，发酵罐产量达15.1 g/L，葡萄糖转化率为20.5 %；通过酶定向挖掘策略获得了有微弱活性的4-O-甲基转移酶，通过分子对接、分子动力学模拟等手段解析了其催化机理和专一性分子机制，理性设计了一系列酶突变体，其中最佳突变体酶的催化活性较野生酶提高了5倍，通过强化辅因子和能量供应，3,3’-二甲氧基鞣花酸的前体物质4-O-甲基没食子酸的摇瓶产量达1 g/L，产量较原始菌株提高14.7倍；通过酶定向挖掘策略挖掘到了高效的过氧化物酶，测定了该酶针对不同底物的酶学动力学参数，优化了该酶的最适表达及催化反应条件。最适条件下，过氧化物酶在体外可催化生成755.79 mg/L 鞣花酸，得率达83%。在此基础上，在大肠杆菌中构建了鞣花酸及3,3’-二甲氧基鞣花酸的生物合成途径，通过过表达hemA提高辅因子血红素供应、优化底物添加量和发酵条件等策略，最终工程菌株合成鞣花酸最高产量达213.55 mg/L，合成3,3’-二甲氧基鞣花酸的最高产量达24.10 mg/L。本项目首次实现了鞣花酸及其衍生物3,3’-二甲氧基鞣花酸的生物合成，为其大规模生产应用奠定理论和技术基础，也为代谢途径未知的天然产物的生物合成提供新思路和新方法。本项目在Nature Communications、Metabolic Engineering及Biotechnology and Bioengineering 等代谢工程及合成生物学领域期刊上发表文章16篇，申请发明专利5件，全面完成了项目预定的研究内容和目标。