大肠杆菌全局转录因子Cra对琥珀酸生物合成过程中CO2固定基因的调控机制

As one of the important four-carbon platform chemicals, succinic acid is classified as the most promising chemicals among 12 bio-based chemicals by U.S. Department of Energy. The CO2 fixed reaction from phosphoenol pyruvate to oxaloacetic acid, which is catalysed by phosphoenolpyruvate (PEP) carboxykinase (PCK) and carboxylase (PPC), has been proved to be the key step for the biosynthesis of succinic acid. The over expression of the pck and ppc genes have been made through the approach of genetic engineering for improving the flux of CO2 fixation, and the improved production of succinic acid was obtained. While the specific cell phenotype is usually regulated by multiple genes and the desired effect is hard to be obtained by simply genetic engineering. In this project, the directed evolution of global transcription factor Cra for further enhancing the production of succinic acid will be proceeded through the transcription level. The direct regulation of Cra on the genes of pck and ppc will be investigated based on the change of the protein structure of Cra in the high yield mutant strain. Finally, based on the global regulation network of CO2 fixation, the molecular mechanism of transcription regulation on the CO2 fixation genes will be studied. For the first time, this project focuses on the global transcription regulation of CO2 fixation genes in order to enhance the succinic acid biosynthesis. The results may be helpful for better understanding and possible regulation of gene expression from the viewpoint of transcription level.

琥珀酸被美国能源部列为12种最具潜力的大宗生物基化学品之首，从磷酸烯醇式丙酮酸(PEP)到草酰乙酸的CO2固定反应是其合成途径的关键步骤，此反应由PEP羧化激酶(PCK)和羧化酶(PPC)催化完成。课题组前期过表达了CO2固定基因pck和ppc，琥珀酸产量有所提高，但由于细胞的特定表型往往由多个基因共同调控，仅对个别基因的改造难以达到预期效果。为了进一步增强琥珀酸合成，本项目拟以大肠杆菌全局转录因子Cra为研究对象，通过对这一转录核心元件结构的理性设计，对代谢网络进行全局性扰动，从而增强内源CO2固定基因pck和ppc表达；然后分析突变前后Cra蛋白结构及功能变化，明晰其转录调控方式，构建全局调控网络，最终阐明Cra在琥珀酸合成过程中调控内源CO2固定基因表达的分子机制。本项目首次利用转录因子全局性调控CO2固定反应，增强琥珀酸生物合成，研究成果预期将为从转录水平调控基因表达提供理论支撑。

琥珀酸被美国能源部列为12种最具潜力的大宗生物基化学品之首，从磷酸烯醇式丙酮酸(PEP)到草酰乙酸的CO2固定反应是其合成途径的关键步骤，此反应由PEP羧化激酶(PCK)和羧化酶(PPC)催化完成。课题组前期过表达了CO2固定基因pck和ppc，琥珀酸产量有所提高，但由于细胞的特定表型往往由多个基因共同调控，仅对个别基因的改造难以达到预期效果。为了进一步增强琥珀酸合成，本项目以大肠杆菌全局转录因子Cra为研究对象，根据转录因子Cra的配体调控域已有的晶体结构信息，利用势能函数计算氨基酸残基取代时的自由能变化，从20个氨基酸中识别关键残基，采用定点突变进行多氨基酸取代，筛选到最优的三点突变方向(D101R+D148R+G274R、D148R+A248F+G274R和N73Y+D148R+G274R)，琥珀酸产量提高了25%以上，实现了Cra蛋白结构的优化。在此基础上，比较Cra突变前后的构象变化及与DNA及配体结合能的变化，确定三点突变后Cra与配体FBP产生了更强的结合力，且能更紧密的与CO2固定的关键基因pck的启动子区域进行识别与结合，从而激活磷酸烯醇式丙酮酸羧化途径。另一方面，以Cra结构域和核糖阻遏蛋白RbsR调控域拼接构成的嵌合体Cra(FFhR)，采用基因芯片技术筛选出FFhR作用下的差异表达基因351个，其中与琥珀酸合成相关基因10个。通过绘制大肠杆菌的代谢网络图谱，并计算FFhR突变产生的代谢流量变化，构建全局调控网络，明确了嵌合体FFhR使胞内代谢流重新分布，进一步激活了CO2固定及乙醛酸支路，从而促进了琥珀酸的生物合成的调控机制。本项目首次利用转录因子全局性调控CO2固定反应，增强琥珀酸生物合成，研究成果将为从转录水平调控基因表达提供理论支撑。