Regulation of Clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of NADH/NAD ratio and ATP pool

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Abstract
Glycerol-glucose-fed (molar ratio of 2) chemostat cultures of Clostridium acetobutylicum were glucose limited but glycerol sufficient and had a high intracellular NADH/NAD ratio (I. Vasconcelos, L. Girbal, and P. Soucaille, J. Bacteriol. 176:1443-1450, 1994). We report here that the glyceraldehyde-3-phosphate dehydrogenase, one of the key enzymes of the glycolytic pathway, is inhibited by high NADH/NAD ratios. Partial substitution of glucose by pyruvate while maintaining glycerol concentration at a constant level allowed a higher consumption of glycerol in steady-state continuous cultures. However, glycerol-sufficient cultures had a constant flux through the glyceraldehyde-3-phosphate dehydrogenase and a constant NADH/NAD ratio. A high substitution of glucose by pyruvate [P/(G+P) value of 0.67 g/g] provided a carbon-limited culture with butanol and butyrate as the major end products. In this alcohologenic culture, the induction of the NADH-dependent butyraldehyde and the ferredoxin-NAD(P) reductases and the higher expression of alcohol dehydrogenases were related to a high NADH/NAD ratio and a low intracellular ATP concentration. In three different steady-state cultures, the in vitro phosphotransbutyrylase and butyrate-kinase activities decreased with the intracellular ATP concentration, suggesting a transcriptional regulation of these two genes, which are arranged in an operon (K. A. Walter, R. V. Nair, R. V. Carry, G. N. Bennett, and E. T. Papoutsakis, Gene 134:107-111, 1993).