Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced byE. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producingE. coli . Deletions ofacrAB androb and, to a lower degree of statistical confidence,emrAB ,mdtEF , andmdtABCD reduced multiple measures of viability, while deletion oftolC nearly abolished FFA production. AnacrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching thetolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in anacrAB + strain, but plasmid-based expression ofacrAB ,mdtEF , and anmdtEF -tolC artificial operon improved the MIC of exogenously added decanoate for anacrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux inE. coli , with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts.