绑定细胞主干途径合成戊二酸和戊二醇的代谢工程研究

In the process of engineering microorganisms to produce chemicals, titer and yield are two key factors determining the feasibility of the pathways. In a metabolic network, compared with that of the central metabolic pathway, the fluxes through the other pathways are relatively low, resulting in the titer and yield unable to meet the practical standard. Facing this general problems, in this project we intend to take the biosynthesis of 1, 5-pentenedioic acid and 1, 5-pentediol as an example, by binding target pathway with central metabolism, coupling product synthesis and cell growth, compelling the cell to readjust the metabolic network, achieving the goal of enhancing pathway flux and effective biosynthesis of target compound. It includes: screening and engineering of candidate enzymes; balancing gene expression by modular optimization; coupling the biosynthetic pathway with central metabolism using pyruvate as the intermediary, the biosynthetic pathway will be coupled with the central metabolism, and investigating the correlation between coupling degree and cell growth/titer; eliminating the bottlenecks through metabolic network analysis and modification; investigating fermentation dynamics and determining the optimal parameters. The final goal is to achieve efficient conversion of pentanedioic acid and pentandiol from inexpensive carbon sources. This study will provide new thought for molecular breeding using synthetic biology, agree with the concept of green development, and has the potential social and economical benefits.

在代谢工程改造微生物合成化学品的过程中，产量和产率是决定途径是否经济可行的关键因素。代谢网络中，相对于中心代谢途径，其他途径的代谢通量较低，导致产量和产率经常难以达到实际应用的要求。面对这一共性问题，本项目拟以1, 5-戊二酸和1, 5-戊二醇的生物合成为例，通过将合成途径与中心代谢途径绑定，实现产物合成与细胞生长偶联，迫使细胞重新调整代谢网络，达到增强目标途径通量和产物高效合成的目的。具体包括：外源酶的筛选及改造；模块化优化平衡基因表达；以丙酮酸为媒介将合成途径与中心代谢偶联，研究偶联程度与细胞生长及产量之间的关系；代谢网络分析及改造消除瓶颈，获得高效合成菌株；发酵动力学研究，确定最佳参数，最终实现戊二酸/戊二醇的高效合成。本研究为利用合成生物学进行分子育种提供新思路，也符合绿色发展的科学理念，具有潜在的社会效益和经济效益。

本项目从酶、途径及网络三个维度对戊二酸/醇等化学品的生物合成开展研究。通过蛋白质工筛选改造提高了关键酶对非天然底物的催化活性；建立了基于莽草酸途径的化学品合成平台，通过新途径设计实现了戊二酸/醇、己二酸等化合物的生物合成。其中戊二酸产量达到54 g/L；通过代谢网络改造，使产物合成与细胞生长耦联，驱动目标产物合成。在Nature Communications等杂志发表SCI论文9篇。