甲醇利用大肠杆菌的构建与调控研究

Methanol is increasingly attractive as a feedstock for biological production to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains. For the bioconversion of methanol to metabolites as biofuels and chemicals, the engineering of microbial platform organism for methanol utilization ability is regarded as a promising method. However, when the engineered methylotrophic Escherichia coli was grown on methanol, the lower tolerance and assimilation ability to intermediate metabolites of formaldehyde were observed. As a result, cell growth was significantly inhibited and methanol metabolism efficiency of the engineered strain was extremely low. In our project, in order to improve the ability of engineered methylotrophic E. coli to utilize methanol, the whole-genome transcript arrays was first applied to analyze the response mechanism of engineered E.coli to formaldehyde to identify key genes associated with formaldehyde tolerance. Subsequently, cell tolerance was enhanced by the regulating the expression of key genes; Second, through identifying promoters that respond to the accumulation of formaldehyde, the dynamic regulation system of the regeneration of formaldehyde acceptor ribulose-5-phosphate by RuMP pathway was established. Subsequently, the interaction between genes, proteins, metabolites and metabolic distribution in this system and methanol metabolism was further investigated. Then, through optimizing the strength and temporal response of the responsive promoters, ribulose-5-phosphate was adequately and timely regenerated during the formaldehyde assimilation process. In our study, by improving the chassis cell tolerance and enhancing formaldehyde assimilation metabolism, we increased the growth ability of engineered E. coli on methanol as a carbon source and developed the methylotrophic E. coli with high methanol utilization ability.

当前,以廉价的甲醇取代粮食作为生物制造的原料来源备受关注。其中，人工改造模式菌株获得甲醇代谢能力是实现甲醇生物转化的有效途径。然而在大肠杆菌中组装甲醇代谢途径时发现，细胞对中间代谢物甲醛表现出较低的耐受和同化能力，导致细胞生长受到抑制且甲醇利用效率极低。本项目以提高大肠杆菌的甲醇利用能力为目标，首先从转录水平解析细胞对甲醛的响应机制，确认甲醛耐受相关的基因，进而通过对关键基因的调节强化底盘细胞的甲醛耐受性能；此外,通过挖掘胞内与甲醛响应的启动子元件，组装RuMP途径，建立甲醛受体5-磷酸核酮糖再生的动态感应调控系统，探明该系统中基因、蛋白、代谢物以及代谢分布与细胞甲醇代谢的作用规律，对关键节点启动子元件的强度和灵敏度进行优化，实现甲醛同化过程中5-磷酸核酮糖适时适量的供给。通过提高底盘细胞的甲醛耐受性以及强化甲醛同化途径，优化细胞以甲醇为碳源生长的能力，获得高效代谢甲醇的重组E.coli。

当前,以廉价的甲醇取代粮食作为生物制造的原料来源备受关注。其中，人工改造模式菌株获得甲醇代谢能力是实现甲醇生物转化的有效途径。然而在大肠杆菌中组装甲醇代谢途径时发现，细胞对中间代谢物甲醛表现出较低的耐受和同化能力，导致细胞生长受到抑制且甲醇利用效率极低。本项目以提高大肠杆菌的甲醇利用能力为目标，首先从转录水平解析细胞对甲醛的响应机制，确认甲醛耐受相关的基因，进而通过对关键基因的调节强化底盘细胞的甲醛耐受性能；此外,通过挖掘胞内与甲醛响应的启动子元件，调控关键代谢物5-磷酸核酮糖的合成，获得高效代谢甲醇的重组E.coli。进而考察了影响甲醇代谢的关键培养条件，并通过重构胞内代谢物的合成以及辅因子途径，实现了甲醇生物转化。