Monday, July 25, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
Growth substrates, specific growth rate (μ) etc. strongly influence Escherichia coli cell physiology. To understand the regulation behind different phenotypic properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). Since biomass composition and carbon balance are rarely measured in the same experiments, possibly leading to distorted MFA results, we carefully carried out both analyses for more accurate quantitative analysis of metabolism. We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor E. coli K-12 MG1655 flux and energy metabolism dynamic responses to change of μ and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing μ and carbon wasting under constant biomass yield. This apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of E. coli pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway. Our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering.