Biosynthetic thiolases catalyze the condensation of two molecules acetyl-CoA to acetoacetyl-CoA and represent key enzymes for carboncarbon bond forming metabolic pathways. An important biotechnological example of such a pathway is the clostridial n-butanol production, comprising various natural constraints that limit titer, yield, and productivity. In this study, the thiolase of Clostridium acetobutylicum, the model organism for solventogenic clostridia, was specifically engineered for reduced sensitivity towards its physiological inhibitor coenzyme A (CoA-SH). A high-throughput screening assay in 96-well microtiter plates was developed employing Escherichia coli as host cells for expression of a mutant thiolase gene library. Screening of this library resulted in the identification of a thiolase derivative with significantly increased activity in the presence of free CoA-SH. This optimized thiolase comprised three amino acid substitutions (R133G, H156N, G222V) and its gene was expressed in C. acetobutylicum ATCC 824 to assess the effect of reduced CoA-SH sensitivity on solvent production. In addition to a clearly delayed ethanol and acetone formation, the ethanol and butanol titers were increased by 46% and 18%, respectively, while the final acetone concentrations were similar to the vector control strain. These results demonstrate that thiolase engineering constitutes a suitable methodology applicable to improve clostridial butanol production, but other biosynthetic pathways involving thiolase-mediated carbon flux limitations might also be subjected to this new metabolic engineering approach. Biotechnol. Bioeng. 2013; 110: 887897. (c) 2012 Wiley Periodicals, Inc.