C. acetobutylicum is a solvent-producing bacterium and produces acetate and butyrate during the initial growth phase which causes a decreased pH value. During transition into the stationary growth phase, the metabolism switches to the production of the solvents acetone, butanol and ethanol. The usability of butanol as biofuel leads to an increasing industrial interest in C. acetobutylicum. To accommodate this fact the intension for this work was to increase the butanol production and to reduce the by-products. The ClosTron® system was used to change this bacterium by genetic manipulation, called “metabolic engineering”. During this work a multitude of insertion mutants has been generated to successively cut off pathways of the complex metabolism. The deactivation of the 3-hydroxybutyryl-CoA dehydrogenase provided an insight into the metabolism without the C4-pathway with the associated butyrate and butanol production. Nevertheless, this strain was still viable, produced spores and a tremendous amount of ethanol. The knock-out of the phosphotransbutyrylase and pH controlled growth conditions changed the organism to a butyrate-negative and high ethanol producing strain. Acetone as a by-product of fermentation could be eliminated by switching off acetoacetyl-CoA:acyl-CoA transferase and was reduced by inactivation of acetoacetat decarboxylase. The creation of double mutants by using the ClosTron® system was performed the first time in C. acetobutylicum and caused a dramatically acetate reduction combined with an acetone-negative phenotype. Recapitulatory, new insights into the function of the different pathways during the solvent production could be gained due to the analysis of the insertion mutants.