In this study, we evaluated the effects of several metabolic engineering strategies in a systematic and combinatorial manner to enhance the free fatty acid (FFA) production in Escherichia coli. The strategies included (i) overexpression of mutant thioesterase I ('TesAR64C) to efficiently release the FFAs from fatty acyl-ACP; (ii) coexpression of global regulatory protein FadR; (iii) heterologous expression of methylmalonyl-CoA carboxyltransferase and phosphoenolpyruvate carboxylase to synthesize fatty acid precursor molecule malonyl-CoA; and (iv) disruption of genes associated with membrane proteins (GusC, MdlA, and EnvR) to improve the cellular state and export the FFAs outside the cell. The synergistic effects of these genetic modifications in strain SBF50 yielded 7.2 ± 0.11 g/L FFAs at the shake flask level. In fed-batch cultivation under nitrogen-limiting conditions, strain SBF50 produced 33.6 ± 0.02 g/L FFAs with a productivity of 0.7 g/L/h from glucose, which is the maximum titer reported in E. coli to date. Combinatorial metabolic engineering approaches can prove to be highly useful for the large-scale production of FA-derived chemicals and fuels.
Keywords: Escherichia coli; FadR; free fatty acids; metabolic engineering; thioesterase.