Metabolic Engineering of Central Carbon Metabolic Pathways

9634569斯蒂芬诺普洛斯这项建议是为了阐明谷氨酸棒杆菌中心碳代谢途径中通量的控制。葡萄糖-6-磷酸、磷酸烯醇丙酮酸和丙酮酸的三个分支点将被特别研究，因为它们被认为是决定合成代谢和分解代谢功能之间的流量分配的关键。将遵循的方法是：(1)分别建立上述分支点的关键酶，如丙酮酸羧基酶、葡萄糖-6-磷酸脱氢酶和磷酸果糖激酶的基本遗传和生化特征，以及(2)根据单个酶的动力学和调节特性重建测量的体内代谢通量。将根据需要进行基因修改，以便在研究中提供适当的遗传背景，或实施特定的通量扩增设计。这项研究将使用最先进的谷氨酸杆菌遗传工具箱，使用物质平衡和放射性碳13化合物标记进行体内代谢通量测定的代谢通量分析，以及设计合理的恒化器实验中的生化分析。代谢网络中通量分配的基本决定因素的研究将通过协调多酶修饰来完成，目的是提高氨基酸生产的生产率和产量，以及在谷氨酸杆菌发酵中过量生产腌制食品风味增强剂海藻糖。***

9634569 Stephanopoulos This proposal is on research to elucidate the control of flux in the central carbon metabolic pathway of Corynebacterium glutamicum. The three branch points of glucose-6-phosphate, phosphoenol-pyruvate and pyruvate will be specifically investigated as they are deemed critical in determining flux distribution between anabolic and catabolic functions. The approach that will be followed is to: (1) establish the fundamental genetic and biochemical characteristics of key enzymes at the above branch points, such as pyruvate carboxylase, glucose-6-phosphate dehydrogenase and phosphofructokinase, respectively, and (2) to reconstruct measured in-vivo metabolic fluxes from the kinetic and regulatory properties of the individual enzymes. Genetic modifications will be employed as needed in order to provide appropriate genetic background in the research, or to implement specific designs for flux amplifications. The research will employ a state of the art genetic toolbox for C. glutamicum, metabolic flux analysis for in-vivo flux determination using material balancing and radioactive carbon thirteen compound labeling, and biochemical assays in properly designed chemostat experiments. The investigation of fundamental determinants of flux distribution in metabolic networks will be concluded with coordinated multienzyme modifications aiming at productivity and yield enhancement of amino acid production, and overproduction of the preserved food flavor enhancer trehalose in C. glutamicum fermentations. ***