The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid beta-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid beta-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid beta-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI(Q) attB-Ptrc-ideal-4-SD phi 10-adhE(Glu568Lys) attB-Ptrc-ideal-4-SD phi 10-atoB attB-Ptrc-ideal-4-SD phi 10-fadB attB-Ptrc-ideal-4-SD phi 10-fadE) synthesises 0.3-1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer.