Several synthetic metabolic pathways for butanol synthesis have been reported in Escherichia coli by modification of the native CoA-dependent pathway from selected Clostridium species. These pathways are all dependent on the O₂ -sensitive AdhE2 enzyme from Clostridium acetobutylicum that catalyzes the sequential reduction of both butyryl-CoA and butyraldehyde. We constructed an O₂ -tolerant butanol pathway based on the activities of an ACP-thioesterase, acting on butyryl-ACP in the native fatty acid biosynthesis pathway, and a promiscuous carboxylic acid reductase. The pathway was genetically optimized by screening a series of bacterial acyl-ACP thioesterases and also by modification of the physical growth parameters. In order to evaluate the potential of the pathway for butanol production, the ACP-dependent butanol pathway was compared with a previously established CoA-dependent pathway. The effect of (1) O₂ -availability, (2) media, and (3) co-expression of aldehyde reductases was evaluated systematically demonstrating varying and contrasting functionality between the ACP- and CoA-dependent pathways. The yield of butanol from the ACP-dependent pathway was stimulated by enhanced O₂ -availability, in contrast to the CoA-dependent pathway, which did not function well under aerobic conditions. Similarly, whilst the CoA-dependent pathway only performed well in complex media, the ACP-dependent pathway was not influenced by the choice of media except in the absence of O₂ . A combination of a thioesterase from Bacteroides fragilis and the aldehyde reductase, ahr, from E. coli resulted in the greatest yield of butanol. A product titer of ∼300 mg/L was obtained in 24 h under optimal batch growth conditions, in most cases exceeding the performance of the reference CoA-pathway when evaluated under equivalent conditions.