Biosynthesis of acetone and n-butanol is naturally restricted to the group of solventogenic clostridia with Clostridium acetobutylicum being the model organism for acetone-butanol-ethanol (ABE) fermentation. According to limited genetic tools, only a few rational metabolic engineering approaches were conducted in the past to improve the production of butanol, an advanced biofuel. In this study, a phosphotransbutyrylase-(Ptb) negative mutant, C. acetobutylicum ptb::int(87), was generated using the ClosTron methodology for targeted gene knock-out and resulted in a distinct butyrate-negative phenotype. The major end products of fermentation experiments without pH control were acetate (3.2 g/l), lactate (4.0 g/l), and butanol (3.4 g/l). The product pattern of the ptb mutant was altered to high ethanol (12.1 g/l) and butanol (8.0 g/l) titers in pH ≥ 5.0-regulated fermentations. Glucose fed-batch cultivation elevated the ethanol concentration to 32.4 g/l, yielding a more than fourfold increased alcohol to acetone ratio as compared to the wildtype. Although butyrate was never detected in cultures of C. acetobutylicum ptb::int(87), the mutant was still capable to take up butyrate when externally added during the late exponential growth phase. These findings suggest that alternative pathways of butyrate re-assimilation exist in C. acetobutylicum, supposably mediated by acetoacetyl-CoA:acyl-CoA transferase and acetoacetate decarboxylase, as well as reverse reactions of butyrate kinase and Ptb with respect to previous studies.