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. ***
