Objectives To establish a technique for efficient fatty acid production through enhancement of coenzyme A (CoA) biosynthesis and malonyl-CoA supply by introducing exogenous pantothenate kinase ( coaA ) and acetyl-CoA carboxylase ( acc ) in Escherichia coli . Results The expression of acc , obtained from Corynebacterium glutamicum , accumulated 2.2-fold more fatty acids in E. coli . The addition of coaA from Pseudomonas putaida or fatty acid synthase ( fasA ) from C. glutamicum resulted in a 3.1- and 3.6-fold increase in fatty acid synthesis in E. coli cells, which expressed acc and coaA , or acc and fasA , respectively. The transformants, simultaneously possessing all three genes, produced 5.6-fold more fatty acids. The strain possessing acc , coaA , and fasA stored 691 mg/L of fatty acids, primarily as phospholipids, inside the inner membrane after 72-h cultivation. In addition, 19% of the total CoA pool was occupied by malonyl-CoA. Conclusions Increased malonyl-CoA significantly contributed to fatty acid production, and the effect was boosted by the expanded total CoA pool. Manipulation of the intracellular CoA species is effective for fatty acid production in E. coli .