Bacteria like Escherichia coli can use propionate as sole carbon and energy source. All pathways for degradation of propionate start with propionyl-CoA. However, pathways of propionyl-CoA synthesis from propionate and their regulation mechanisms have not been carefully examined in E. coli. In this study, roles of the acetyl-CoA synthetase encoding gene acs and the NAD(+-)dependent protein deacetylase encoding gene cobB on propionate utilization in E. coil were investigated. Results from biochemical analysis showed that, reversible acetylation also modulates the propionyl-CoA synthetase activity of Acs. Subsequent genetic analysis revealed that, deletion of acs in E. coli results in blockage of propionate utilization, suggesting that acs is essential for propionate utilization in E. coil. Besides, deletion of cobB in E. coli also results in growth defect, but only under lower concentrations of propionate (5 mM and 10 mM propionate), suggesting the existence of other propionyl-CoA synthesis pathways. In combination with previous observations, our data implies that, for propionate utilization in E. coil, a primary amount of propionyl-CoA seems to be required, which is synthesized by Acs. (C) 2014 Elsevier Inc. All rights reserved.