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

Abstract

The levels of seven intermediary enzymes involved in acetate and butyrate formation from acetyl coenzyme A in the saccharolytic anaerobe Clostridium acetobutylicum were investigated as a function of time in solvent-producing batch fermentations. Phosphate acetyltransferase and acetate kinase, which are known to form acetate from acetyl coenzyme A, both showed a decrease in specific activity when the organism reached the solvent formation stage. The three consecutive enzymes thiolase, P-hydroxybutyrylcoenzyme A dehydrogenase, and crotonase exhibited a coordinate expression and a maximal activity after growth had ceased. Only low levels of butyryl coenzyme A dehydrogenase activity were found. Phosphate butyryltransferase activity rapidly decreased after 20 h from 5 to 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 2.5to 10-fold increase in specific activity after phosphate butyryltransferase activity no longer could be detected. These results suggest that 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. This indicates that none of the maximal activities of the enzymes assayed is rate limiting in C. acetobutylicum.

Spo0A directly controls the switch from acid to solvent production in solvent‐forming clostridia

“…There have been conflicting reports concerning the pattern of phosphotransbutyrylase activity during the bacterial growth cycle (Hartmanis and Gatenbeck, 1984;Palosaari and Rogers, 1988;Papoutsakis and Bennett, 1993). Most recently, Tummala et al (1999) showed that ptb expression in C. acetobutylicum is associated with the early growth phase, decreasing steadily after late exponential phase, and that it corresponds closely to the activity of phosphotransbutyrylase in crude cell extracts.…”

Structure and transcription of genes within the β-hbd-adh1 region of Clostridium acetobutylicum P262

“…Expression of the P-hbd and ORFB genes was similar in that both genes were transcribed during the acidogenic, pH breakpoint and solventogenic phases, and were not induced or derepressed prior to solventogenesis. Transcription of the P-hbd gene is to be expected during the acidogenic and solventogenic phase,s, since the central fermentation pathway is involved in the production of both butyric acid and butanol [l], and the BHBD enzyme was shown to peak at 17 h by Hartmanis and Gatenbeck [16]. However, the pattern of expression of the adhl gene contrasted with the transcription of the P-hbd and ORFB genes in that the adhl gene was induced or derepressed prior to solventogenesis.…”

Regulation of nitrogen metabolism, starch utilisation and the β-hbd—adh1 gene cluster in Clostridium acetobutylicum

“…The fl-hbd and fixB genes were both transcribed throughout the acidogenic, pH breakpoint and solventogenic phases, and were not induced or darepressed during solventogenesis. Transcription of the fl-hbd gene is to be expected during the acidogenic and solventogenic phases, since the central fermentation pathway is involved in the production of both butyric acid and butanol [1], and the BHBD enzyme was shown by Hartmanis and Gatenbeck [20] to peak at 17 h. However, the pattern of expression of the adhl gene contrasted with the transcription of the fl-hbd and ftxB genes in that the adhl gene was induced or derepressed prior to solventogenesis. This differential expression of the adhl gene indicated that it was independently expressed on a 2.4-kb transcript and peaked at 12 h, which was similar to the time of expression of other genes involved in solvent production [21,22].…”