脂肪酸非天然生物转化酶系的分子工程及组合催化研究

The sustainability issue of chemical industry has to be considered seriously due to the crisis of resources shortage and environmental pollution caused by excessive consumption of fossil resources. The strategy to address this problem is either to replace the fossil feedstocks with renewable raw materials or to replace the traditional chemo-catalytic or non-catalytic reactions with biocatalytic processes. Based on the bio-originated fatty acids as starting materials, this project proposes first to construct a library of various enzyme parts required for unnatural biotransformation of fatty acids by making use of the huge genome data and powerful enzyme-molecular engineering approaches, and then to improve their catalytic performance by rational design or directed evolution. Subsequently, the modules required to biosynthesize omega-hydroxyl fatty acids, alpha, omega-dicarboxylic acids or omega-amino fatty acids are constructed by assembling different enzyme parts needed. The compatibility, cooperativity and stability of the multi-enzyme modules have to be assessed scientifically to identify those problematic parts or modules that need to be substituted or engineered for improving efficiency and/or working period. Finally, the engineering issues like the efficiency and stability of multi-enzyme modules in reactors will be addressed by bioreaction engineering approaches. The research achievements will provide scientific guidance, technical solution and bioresourcing support for the biosynthesis of biodegradable polymers and are expected to be of importance for the development of China’s synthetic biotechnology and bioindustries.

化石资源过度消耗所引发的资源和环境危机，促使科学家不得不考虑化学工业的可持续性问题，主要对策包括使用可再生原料替代化石原料，或采用生物催化工艺替代传统催化或非催化反应工艺。本课题拟以生物基脂肪酸为出发原料，利用庞大的基因组数据资源和日趋成熟的酶分子工程改造技术，构建脂肪酸非天然生物转化所需的各种酶元件库，并进行理性设计和定向进化以提高催化性能；然后将不同的酶元件组装在一块，构建生物合成羟基酸、二元酸和氨基羧酸等聚酯/聚酰胺单体所需的酶模块库，科学评价多酶模块的相容性、协同性和稳定性，并对有问题的元件或模块进行替换或改造，以提高多酶模块的效率和寿命；最后通过生物反应工程方法解决多酶模块在反应器中运行的效率和稳定性等工程问题。预期研究成果可为生物合成可降解高分子材料提供科学指导、技术方案和生物资源保障，对发展我国的合成生物工程学科和产业也将具有重要意义。

设计、建立了具有我国自主知识产权的脂肪酸非天然生物转化人工酶元件和组件库；完成了系列中链双官能团化学品的途径设计、元件挖掘、模块组装和系统集成；通过体外多酶级联，成功合成获得了辛二酸/壬二酸/月桂二酸、8-羟基辛酸/8-氨基辛酸、以及9-羟基壬酸/9-氨基壬酸等高附加值多功能化学品，为基于油酸等非石化原料绿色合成生态友好的化学品或材料单体开拓了新的方向和途径。其中，本项目发现的具有非常规(Abnormal)位置选择性的Baeyer-Villiger单加氧酶等关键元件和发明的即插即用、灵活组装型催化模块(酶组件)具有非常显著的创新性和实用性。本项目所建立的新酶资源、关键技术以及人才团队，也为近期获批国家合成生物学专项项目(No. 2019YFA09005000: 新分子生化反应设计与生物合成系统创建)奠定了坚实基础。