枯草芽孢杆菌中母乳寡糖代谢网络的模块优化与动态调控

微生物代谢工程已成为工业微生物育种和细胞工厂构建的重要手段。但是，传统代谢工程方法对复杂代谢途径的调控效果往往不佳。如何对复杂代谢途径进行高效、精准调控是代谢工程领域的一个关键难题。本项目以典型的模式工业微生物和食品安全菌—枯草芽孢杆菌(Bacillus subtilis)为研究对象，以重要的功能营养品—母乳寡糖(Human milk oligosaccharides, HMOs)为目标产物，进行代谢网络模块动态调控体系的设计与构建。通过双碳源高效利用系统的构建、基因精准表达调控元件的挖掘、动态调控方法的设计，以及代谢网络的模块组装，实现代谢网络的全局优化和动态平衡。本项目拟采用的方法将有助于解决代谢工程中存在的复杂代谢途径调控效率低的难题，对微生物代谢精准调控将起到积极的促进作用，同时对高效构建B. subtilis细胞工厂合成重要功能营养品具有重要借鉴作用。

Metabolic engineering is an enabling technology for developing microbial cell factories to produce industrially important and pharmaceutically useful biochemicals in green and sustainable manners. However, the complexity of metabolic pathway and regulatory network reduce the efficiency of metabolic engineering for construction of efficient production host. In this project, we focus on how to solve the key issues on metabolic engineering for effectively and precisely regulating complex metabolic pathway and regulatory network with biosynthesis of human milk oligosaccharides (HMOs) pathway in Bacillus subtilis as an example. A dynamically modular pathway engineering approach for metabolic network optimization is expected to be developed as a generic strategy for metabolic engineering. Specifically, we firstly plan to construct glucose and lactose co-utilization pathways to supply sufficient carbon moiety for HMOs synthesis. Next, encoding genes of key enzymes in HMOs synthetic pathway will be assembled with regulatory expression elements, such as various ribosome binding site sequences and promoters, to precisely fine-tune HMOs synthetic pathway. Next, quorum-sensing promoter, ligand-bound nucleic acid aptamer transcriptional regulatory method, and clustered regularly interspaced short palindromic repeats interference (CRISPRi) system will be optimized and used to dynamically regulate HMOs synthetic modules, GDP-fucose synthetic module, central carbon metabolic module, and cofactor supply module. Finally, global and dynamical module optimization strategies will be investigated to unraveling interaction mechanism between carbon metabolism and cofactor metabolism, fine-tuning key precursor supply and cofactor supply, and realizing global optimization and dynamic balance of metabolic network. The dynamically modular pathway engineering approach for metabolic network optimization will be a useful and generally applicable strategy for fine-tuning complex metabolic pathway and network, which will facilitate the improvement of efficiency of metabolic engineering for microbial cell factories construction.