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) inEscherichia coli. Results The expression ofacc, obtained fromCorynebacterium glutamicum, accumulated 2.2-fold more fatty acids inE. coli. The addition ofcoaAfromPseudomonas putaidaor fatty acid synthase (fasA) fromC. glutamicumresulted in a 3.1- and 3.6-fold increase in fatty acid synthesis inE. colicells, which expressedaccandcoaA, oraccandfasA, respectively. The transformants, simultaneously possessing all three genes, produced 5.6-fold more fatty acids. The strain possessingacc,coaA, andfasAstored 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 inE. coli.