丙酮酸激酶PykA调控厌氧酵解速率的机理研究

Glycolysis is the most important pathway for glucose catabolism. For anaerobic fermentation system, glycolysis not only provides necessary precursors for cell growth and metabolism, but also produces most ATP needed for life through substrate level phosphorylation. Thus, enhancing glycolysis is of vital importance for the application of industrial microbes. In our previous study, we have observed a surprising result, that the inactivation of pyruvate kinase PykA of Escherichia coli significantly improves the anaerobic growth and the glucose utilization. Moreover, we found that PykA has a very high affinity and catalytic efficiency toward ADP, which might compete with the ADP-dependent 3-phosphoglycerate kinase for ADP, thereby reduce the glycolytic rate under anaerobic conditions. Based on this hypothesis, the mechanism of how PykA regulates the anaerobic glycolytic rate will be investigated in the project. Considering the high affinity of PykA toward ADP, enzymatic kinetics of 3-phosphoglycerate kinase, gene expression regulation of PykA and comparative transcriptional analysis will be examined to investigate the relationship among the expression level of the pykA gene, the ADP availability, the reaction rate of the ADP-dependent 3-phosphoglycerate kinase, and the anaerobic glycolytic rate of E. coli. Novel mechanisms for controlling the glycolysis and regulating the glycolytic rate under anaerobic conditions are to be discovered, with the aim to develop novel strategies for increasing the glycolytic rate and creating super glycolytic pathway.

酵解途径是自然界中最重要的糖代谢途径。对厌氧发酵体系而言，酵解途径不仅为生长和代谢提供必要的前体物质，还是微生物获得能量的主要途径，因此提高酵解速率对工业微生物的应用具有重要意义。我们在前期研究中观察到一个新现象：丙酮酸激酶PykA失活后，大肠杆菌厌氧生长和葡萄糖代谢能力反而提高了。进一步研究发现，PykA对ADP具有非常高的亲和力和催化效率。由此推测，PykA会与酵解途径中同样以ADP为辅因子的磷酸甘油酸激酶PGK竞争ADP，从而降低酵解速率。为了研究该假设是否成立，本研究拟从PykA对ADP的高亲和力出发，分别从酶促反应动力学、基因表达调控、转录调控的角度，研究“pykA基因表达量-ADP分配-磷酸甘油酸激酶反应速率-糖酵解速率”之间的关系，发现影响糖酵解速率的新控制点，揭示厌氧条件下酵解途径调控的新机制，建立调控酵解速率的新策略，为创建高速酵解途径提供新的理论依据。

酵解途径是自然界中最重要的糖代谢途径。对厌氧发酵体系而言，酵解途径不仅为生长和代谢提供必要的前体物质，还是微生物获得能量的主要途径，因此提高酵解速率对工业微生物的应用具有重要意义。我们在前期研究中观察到一个新现象：丙酮酸激酶PykA失活后，大肠杆菌厌氧生长和葡萄糖代谢能力反而提高了。进一步研究发现，PykA对ADP具有非常高的亲和力和催化效率。由此推测，PykA会与酵解途径中同样以ADP为辅因子的磷酸甘油酸激酶Pgk竞争ADP，从而降低酵解速率。为了研究该假设是否成立，本研究从PykA对ADP的高亲和力出发，建立了PykA、PykF和磷酸甘油酸激酶Pgk的酶促反应动力学模型，研究了底物ADP充足和限制条件下各酶反应动力学模型，阐明了PykA在ADP限制条件下与糖酵解途径其他酶（如本研究的磷酸甘油酸激酶Pgk）竞争ADP，从而影响整个糖酵解途径，为PykA调控大肠杆菌厌氧代谢提供数据支撑。继而发现在大肠杆菌中PykA及糖转运模块均是调控糖消耗及酵解途径的关键限制因素，过量表达调控PykA不能提高厌氧糖酵解速率，但敲除pykA基因结合强化糖转运模块，可以提高底物糖消耗速率，建立了敲除pykA基因并强化糖转运模块以促进糖酵解策略。另外，发现了与PykA匹配性更好的磷酸甘油酸激酶hPgk，为创建高速酵解途径奠定了基础。