In Vivo NMR Analysis of the Influence of Pyruvate Decarboxylase and Alcohol Dehydrogenase of Zymomonas mobilis on the Anaerobic Metabolism of Escherichia coli

Abstract

AbstractPhosphorus‐31 NMR studies of Escherichia coli expressing cloned pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) genes from Zymomonas mobilis revealed significant changes in the concentration of intracellular metabolites. The intracellular concentrations of phosphate, sugar phosphates, NAD(H), NTP, NDP, and UDPG of the strain HB101 expressing pdc, adh, or both genes were analyzed at quasi steady state during the anaerobic catabolism of glucose. The evolution of the intracellular pH and extracellular pH during glucose consumption showed a constant value of ΔpH equal to 0.2 pH unit that did not depend on the expression of the Zymomonas genes. The strains expressing PDC only exhibited greatest changes in intracellular composition with significantly decreased inorganic phosphate, NAD(H), NTP, and UDPG and significantly increased sugar phosphates. Analysis of the fermentation products during the NMR experiments provided further information about the physilogical changes observed in the cells. Two plasmid effects on the host metabolism are reported, one induced by the plasmid utilized as control (pUCl9), and the other induced by the expression of the pet operon. In both cases an enhancement of the glucose uptake rate was observed. Although the plasmid carrying adh induces a rapid metabolic inhibition, the expression of pdc does not. The expression of pdc and adh together showed a physiological enhancement that is attributable not to effects on external pH but rather to the effect of the switch in pathways from mixed acid to ethanol fermentation.