甲醇生物合成异戊二烯途径工程研究

With the development of synthetic biology, it becomes possible to construct cell factories by rational design and reconstruct of novel synthetic pathways based on the properties of feedstocks and/or products. The present project aims at constructing a novel pathway converting methanol to isoprene with methanol-utilizing bacteria Methylobacterium extorquens AM1 as chassis. The difficulty of this work lies in the lack of genetic tools in M.extorquens AM1, and the flux balancing of multi-gene pathways. Inspired by the idea of modular optimization, the isoprene synthesis pathways were proposed to separate into two modules.The upstream-module will be integrated into the genome of M.extorquens AM1, and the downstream-module will be introduced by heterogeneous pathways. After the precise balancing and optimizing the fluxes of these two modules, a recombinant strain with high yield of isoprene will be acquired. With further optimization of the fermentation processes for biosynthesis isoprene from methanol, a novel technology with independent intellectual property rights is supposed to be established. This Methanol-based isoprene route will potentially competes with sugar-based route as it will extends the value-added chain of coal chemical industries, and potentially be a new direction connecting industrial biotechnology and methanol-based chemical industry.

合成生物学对生物化工的学科发展具有重要推动作用，可以根据原料特点和产物合成需求，通过全新的代谢途经设计和重构，构建高效的细胞工厂。本项目以扭脱甲基杆菌为底盘，构建以甲醇为原料高效生物合成异戊二烯的新途径。为克服底盘微生物基因操作工具缺乏，多基因途径代谢流匹配复杂的难题，本项目提出模块化途径重构的思路，利用底盘微生物改造和外源途径重构相结合的策略，将异戊二烯合成途径分为上下两个模块，分别从上游模块整合与底盘优化、下游模块重构与代谢流精细匹配以及菌株培养与异戊二烯生产动力学特性三个方面进行研究，以期建立具有自主知识产权的甲醇高效转化异戊二烯的细胞工厂及新工艺。利用生物法高效转化甲醇生成异戊二烯，具有与糖基生物异戊二烯路线竞争的优势，突破国际公司专利封锁；同时从我国国情出发，延伸和增值煤化工产业链，将成为工业生物技术与甲醇化工结合的新方向。

合成生物学对生物化工的学科发展具有重要推动作用，可以根据原料特点和产物合成需求，通过全新的代谢途经设计和重构，构建高效的细胞工厂。微生物发酵的碳源是决定化学品生物制造经济性的关键因素。甲醇来源广泛，以甲醇作为碳源能够有效缓解糖基生物制造“与人争粮”及原料经济性等问题。本项目以扭脱甲基杆菌外源合成甲羟戊酸为模式体系，为解决其缺乏兼容性质粒和循环代谢网络调控手段不足的问题，通过基因组途径工程实现稳定合成甲羟戊酸，通过QscR调控蛋白工程改造循环代谢网络；进一步通过培养工艺优化解决菌株生长不稳定、细胞中还原力不足及甲醇耐受性低等关键问题，系统研究扭脱甲基杆菌生物合成过程的强化方法，以期为建立甲羟戊酸下游对接萜类化合物的合成途径工程提供基础。通过上述努力，成功构建了以甲醇为底物生产甲羟戊酸的工程菌，所构建的甲醇高效转化甲羟戊酸重组菌株，转化率达到理论转化率的10%，总体产量达到4.35g/L，为公开文献报道最高水平。