The current study aimed to construct a functionally reversed beta-oxidation cycle in Escherichia coil for the production of adipic acid, one of the most important dicarboxylic acids. Starting from the condensation of acetyl-CoA and succinyl-CoA, a synthetic pathway mimicking a reversed beta-oxidation cycle was constructed in E. coil to produce adipyl-CoA, consisting of four enzymatic steps. Products of the synthetic pathway were confirmed by liquid chromatography-tandem mass spectrometry. To convert adipyl-CoA into adipic acid, a putative adipyl-CoA thioesterase (encoded by Acot8 from Mus musculus) was selected, and its functional expression in E. coil was confirmed by gas chromatography-mass spectrometry. Finally, when the reversed beta-oxidation cycle and Acot8 were co-expressed in E. coil, the resulting strain produced 12 mu g/L of adipic acid from glucose. These results suggest that the synthetic adipic acid pathway functions in E. coil but requires further optimization for increased production of adipic acid. (C) 2015 Elsevier Ltd. All rights reserved.