This study investigated the effect of the methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii on production of free fatty acid (FFA) in Escherichia coli. Overexpression of the MMC exhibited a 44% increase in FFA titer. Co-overexpression of MMC and phosphoenolpyruvate carboxylase (PPC), which supplies the MMC precursor, further improved the titer by 40%. Expression of malic enzyme (MaeB) led to a 23% increase in FFA titer in the acetyl-CoA carboxylase (ACC)-overexpressing cells, but no increase in the MMC-overexpressing cells. The highest FFA production in the MMC-overexpressing strain was achieved through the addition of aspartic acid, which can be converted into oxaloacetate (OAA), resulting in a 120% increased titer compared with that in the ACC-overexpressing strain. These findings demonstrate that MMC provides an alternative pathway for malonyl-CoA synthesis and increases fatty acid production. (C) 2017 Elsevier Ltd. All rights reserved.