The physiological changes in an F 1 -ATPase-defective mutant of Escherichia coli W1485 growing in a glucoselimited chemostat included a decreased growth yield (60%) and increased specific rates of both glucose consumption (168%) and respiration (171%). Flux analysis revealed that the mutant showed approximately twice as much flow in glycolysis but only an 18% increase in the tricarboxylic acid (TCA) cycle, owing to the excretion of acetate, where most of the increased glycolytic flux was directed. Genetic and biochemical analyses of the mutant revealed the downregulation of many TCA cycle enzymes, including citrate synthase, and the upregulation of the pyruvate dehydrogenase complex in both transcription and enzyme activities. These changes seemed to contribute to acetate excretion in the mutant. No transcriptional changes were observed in the glycolytic enzymes, despite the enhanced glycolysis. The most significant alterations were found in the respiratory-chain components. The total activity of NADH dehydrogenases (NDHs) and terminal oxidases increased about twofold in the mutant, which accounted for its higher respiration rate. These changes arose primarily from the increased (3.7-fold) enzyme activity of NDH-2 and an increased amount of cytochrome bd in the mutant. Transcriptional upregulation appeared to be involved in these phenomena. As NDH-2 cannot generate an electrochemical gradient of protons and as cytochrome bd is inferior to cytochrome bo 3 in this ability, the mutant was able to recycle NADH at a higher rate than the parent and avoid generating an excess proton-motive force. We discuss the physiological benefits of the alterations in the mutant.The elucidation of the regulatory mechanism of glycolytic flux is critical for the development of effective fermentation processes for the production of useful metabolites by microorganisms. Glycolytic flux in Escherichia coli is controlled primarily by the ATP demand of the cells rather than by glycolytic enzymes (22). For example, defects in the activity of F 1 F o -ATP synthase that impair oxidative phosphorylation (21,38,40) or increased ATPase activity in hydrolyzing ATP (22), which both lead to a reduced ATP/ADP ratio (21, 22), result in enhanced rates of glucose consumption. The enhancement of glucose consumption by defective F 1 F o -ATP synthase activity has also been reported for the gram-positive bacteria Bacillus subtilis (32) and Corynebacterium glutamicum (34), which are industrially important. Several attempts have been made to apply these findings to the production of useful metabolites from glucose by fermentation. Our group reported the first successful application of pyruvate production, using an E. coli mutant with a defective F 1 -ATPase (40). In this case, enhanced pyruvate production was achieved with an increased rate of glucose consumption. The effectiveness of F 1 F o -ATP synthase defects for the production of acetate (9), as well as pyruvate (8), has also been reported for different E. coli mutants. Recently, we demonstrated that...
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