Metabolic Flux Map of E. Coli ptsG Mutant and Wild Type Consuming Glucose/Xylose under Anaerobic Condition Lignocellulose derived from agricultural residue is a potential low cost feed stock for the production of both for fuels and industrial chemicals. The hydrolysis of lignocelluloses yields a mixture of sugars containing mainly glucose, arabinose and xylose.  In the presence of glucose, E. coli shows sequential sugar consumption when it is grown in media derived from lignocelluloses hydrolyzates. The simultaneous consumption of sugars in a mixture would be advantageous in fermentative production process as it would eliminate diauxic growth, therefore reducing operating time and increasing productivity. The carbon catabolite repression phenomenon is a result of a sophisticated sugar utilization regulatory system (SURS) which modulates sugar consumption and the direction of carbon flow inside the cell.

 The E. coli SURS consist of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS) and several global regulators including CRP (cAMP receptor protein), Mlc (controlling several glycolytic, gluconeogenic and glucose-related genes), and Cra (catabolite repressor/activator protein).  Our laboratory is involved in the comprehensive evaluation of E. coli SURS. The Gonzalez group has constructed E. coli strain devoid of gene encoding ptsG. The ptsG is involved in the transport of glucose and plays an important role in carbon catabolite repression. The ptsG- strain doesn't show diauxic growth and consumes glucose and xylose simultaneously. However, it grows slower than wild type grown on glucose and has a long lag time. We are currently doing more comprehensive study E. coli SURS by conventional metabolic flux analysis that is based on metabolite balance and extra cellular measurement. It is a useful tool for the first pass analysis but more rigorous analysis usually is done by ¹³C labeling based MFA. Each labeling measurement dependent on the intracellular flux poses additional constraints on the set of intracellular fluxes. However, the ¹³C labeling based MFA has been used for aerobic system and has not done been for anaerobic system. The ¹³C MFA is challenging under anaerobic condition as there is not less rearrangement because TCA cycle is not complete. The information from the labeled substrate can be increased through the proper choice of labeled substrate. We have come up with the optimal choice of labeled substrate for the experimental condition by flux indentifiablity analysis. The metabolic network model for MFA has been constructed for E. coli and it consists of all principal pathway of primary metabolism (PTS transport of glucose, glycolysis, pentose phosphate pathway, TCA cycle, glyoxylate shunt, and fermentative reactions) and the biosynthetic pathways that convert  the primary metabolic precursors to sink metabolites. The results of ¹³C metabolic flux analysis of E. coli ptsG mutant and wild type will be presented.