Metabolic Engineering of Central Carbon Metabolic Pathways

该提案是关于研究阐明谷氨酸棒杆菌的中心碳代谢途径中的通量控制。 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，因为它们被认为在确定合成代谢和分解代谢功能之间的通量分布中至关重要。 将遵循的方法是：（1）建立在上述分支点的关键酶，如丙酮酸羧化酶，葡萄糖-6-磷酸脱氢酶和磷酸果糖激酶，分别的基本遗传和生化特性，和（2）重建测量的体内代谢通量从单个酶的动力学和调节特性。 将根据需要进行遗传修饰，以便在研究中提供适当的遗传背景，或实施通量扩增的特定设计。 这项研究将采用最先进的遗传工具箱为C。谷氨酸的代谢通量分析，使用物质平衡和放射性碳十三化合物标记的体内通量测定的代谢通量分析，以及在适当设计的恒化器实验中的生物化学测定。 研究代谢网络中通量分布的基本决定因素将通过协调的多酶修饰来结束，目的是提高氨基酸生产的生产率和产量，以及在C. 谷氨酸发酵。 ***

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. ***