REGULATION OF CLOSTRIDIUM-ACETOBUTYLICUM METABOLISM AS REVEALED BY MIXED-SUBSTRATE STEADY-STATE CONTINUOUS CULTURES - ROLE OF NADH NAD RATIO AND ATP POOL/

Glycerol-glucose-fed (molar ratio of 2) chemostat cultures of Clostrid ium acetobutylicum were glucose limited but glycerol sufficient and ha d 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 o f the glycolytic pathway, is inhibited by high NADH/NAD ratios. Partia l substitution of glucose by pyruvate while maintaining glycerol conce ntration at a constant level allowed a higher consumption of glycerol in steady-state continuous cultures. However, glycerol-sufficient cult ures had a constant flux through the glyceraldehyde-3-phosphate dehydr ogenase 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 cult ure with butanol and butyrate as the major end products. In this alcoh ologenic culture, the induction of the NADH-dependent butyraldehyde an d the ferredoxin-NAD(P) reductases and the higher expression of alcoho l dehydrogenases were related to a high NADH/NAD ratio and a low intra cellular ATP concentration. In three different steady-state cultures, the in vitro phosphotransbutyrylase and butyrate-kinase activities dec reased with the intracellular ATP concentration, suggesting a transcri ptional regulation of these Cove genes, which are arranged in an opero n