内源电子介体介导的大肠杆菌电合成丁二酸的途径构建与调控研究

As the excellent biocompatibility and electron transfer efficiency of endogenous electron shuttle of electrogenic microorganism, it has wide application potential in microbial electrosynthesis chemical. However, in the model organism such as E.coli, the transfer efficiency of endogenous electron into cells was low due to the lack of specific porin protein for endogenous electron shuttle. Meanwhile, the unbalance of intracellular proton motive force was caused by the uptake of exogenous electrons, resulting in the metabolic disturbance in vivo. The objective of our project is to construct endogenous electron shuttle phenazine-1-carboxylic acid electron transport pathway for efficient electrosynthesis of succinic acid in E.coli. First, in order to decrease the electron transport resistance across the cell membrane and increase the extracellular electron transport efficiency, specific porin protein for electron shuttle is assembled to construct endogenous electron shuttle transmembrane transport channels in E.coli . Subsequently, the interaction between changes in extracellular electron transport pathways and intracellular ATP levels would be analyzed. On this basis, a dynamic ATP regeneration system is constructed to maintain intracellular ATP levels, strengthen cellular physiological metabolism and increase intracellular electron utilization efficiency. Finally, a recombinant E.coli with high-efficiency electrosynthesis using endogenous electron shuttle is obteain. The electrosynthesis conditions would be optimized to achieve efficient electrosynthesis of succinic acid by E.coli. The results of this project can provide insights for microbial electrosynthesis chemicals.

产电微生物中的内源电子介体由于具有优良的生物相容性与电子传递效率，在微生物电合成化学品方面具有广阔的应用潜力。然而，大肠杆菌等模式生物由于缺乏内源电子介体的特异性通道蛋白导致外源电子较高的传质阻力，而且细胞对外源电子的摄取打破了胞内质子动力势平衡，引起细胞生理代谢紊乱，限制了内源电子介体介导的电合成效率。本项目以在E.coli中构建内源电子介体吩嗪-1-羧酸电子传递途径高效电合成丁二酸为目标，首先通过在E.coli中组装电子介体特异性通道蛋白，搭建内源电子介体跨膜转运通道，降低细胞膜电子传递阻力，提高胞外电子传递速率；其次，解析胞外电子传递途径变化与胞内ATP水平的相互作用关系，构建ATP动态感应再生系统，维持胞内ATP水平，强化细胞生理代谢功能，提高胞内电子利用效率。最终构建出利用内源电子介体高效电合成的重组大肠杆菌，结合电合成条件优化，实现E.coli高效电合成丁二酸。

产电微生物中的内源电子介体由于具有优良的生物相容性与电子传递效率，在微生物电合成化学品方面具有广阔的应用潜力。然而，大肠杆菌等模式生物由于缺乏内源电子介体的特异性通道蛋白导致外源电子较高的传质阻力，而且细胞对外源电子的摄取打破了胞内质子动力势平衡，引起细胞生理代谢紊乱，限制了内源电子介体介导的电合成效率。.本项目首先筛选来源于铜绿假单胞菌和大肠杆菌的孔道蛋白，在E. coli搭建内源电子介体跨膜转运通道，PCA的胞外含量提高了20.5%，降低了细胞膜对电子介体跨膜的阻力；其次，分析电子传递途径变化与胞内ATP水平的相互作用关系，构建yadO核糖开关，实现了yadO核糖开关响应胞内ATP的水平调控下游基因的表达并进一步通过胞外磷酸盐浓度调控胞内ATP水平；最后，组装ATP动态感应再生系统，维持胞内ATP水平，强化细胞生理代谢功能，并应用于内源电子介体电合成体系中，通过电化学分析显示强化了氧化还原反应，最终电合成丁二酸水平提高17.9%。本项目的研究成果为大肠杆菌高效利用电子合成还原性代谢产物的设计与过程控制提供思路与借鉴，为将电能向化学能转化的实践提供理论基础。