Intermediary Metabolism in Clostridium acetobutylicum : Levels of Enzymes Involved in the Formation of Acetate and Butyrate

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Abstract
The levels of 7 intermediary enzymes involved in acetate and butyrate formation from acetyl CoA in the saccharolytic anaerobe C. acetobutylicum were investigated as a function of time in solvent-producing batch fermentations. Phosphate acetyltransferase and acetate kinase, which form acetate from acetyl CoA, both showed a decrease in specific activity when the organism reached the solvent formation stage. The 3 consecutive enzymes thiolase, .beta.-hydroxybutyryl CoA dehydrogenase and crotonase exhibited a coordinate expression and a maximal activity after growth had ceased. Only low levels of butyryl CoA dehydrogenase activity were found. Phosphate butyryltransferase activity rapidly decreased after 20 h from 5-11 U/mg of protein to below the detection limit (1 mU/mg). Butyrate no longer can be formed and the metabolic flux may be diverted to butanol. Butyrate kinase showed a 2.5- to 10-fold increase in specific activity after phosphate butyryltransferase activity no longer could be detected. Thus, the uptake of acetate and butyrate during solvent formation can not proceed via a complete reversal of the phosphate transferase and kinase reactions. The activities of all enzymes investigated as a function of time in vitro are much higher than the metabolic fluxes through them in vivo, indicating that none of the maximal activities of the enzymes assayed is rate limiting in C. acetobutylicum.