From a potential acetyl-CoA synthetase gene (acs) predicted from E. coli genome sequencing, we created an insertion mutation of acs on the chromosme. The acs mutant has a deficiency in utilizing acetate especially at low concentration, although the strain still metabolizes acetate via Pta/AckA pathway. When monitored by cat fusion to the acs promoter, the repression by glucose was observed while the removal of negative regulators for glyoxylate bypass, iclR and fadR, did not affect the expression of acs. It was also found that the FruR, a global regulator, acts as a positive regulator for the acs gene.