Introduction of an Acetyl-CoA Carboxylation Bypass into Escherichia coli for Enhanced Free Fatty Acid Production

Abstract

Microbial production of free fatty acids (FFAs) and their derivatives from renewable biomass is considered a promising approach to replace petroleum-based products. For the reason, redesigning microbes has been made for increased fatty acid production through metabolic engineering and synthetic biology. Here, we have expressed methylmalonyl-CoA carboxyltransferase (MMC) genes from Propionibacterium freudenreichii shermanii as a key manipulation for an acetyl-CoA carboxylation bypass in Escherichia coli. The resulting strain increased free fatty acid (FFA) production by 44%. Co-expression of MMC and phosphoenolpyruvate carboxylase (PPC) to supply oxaloacetate (OAA), the MMC precursor, further elevated FFA titer by 40%. Unlike overexpression of acetyl-CoA carboxylase (ACC), overexpression of MMC redirects carbon flux from the tricarboxylic acid (TCA) cycle to the fatty acid synthetic pathway. The addition of aspartic acid, converted into OAA, exhibited the highest FFA production titer, a 120% increased titer compared with that in the ACC overexpressing strain. These findings demonstrate that MMC bypass could provide an alternative pathway for malonyl-CoA synthesis and increases fatty acid production.