Escherichia coli has been used as platform host for studying the production of free fatty acids (FFA) and other energy dense compounds useful in biofuel applications. A majority of FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet proteins that may be responsible for export remain unknown. 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 fifteen genes and operons were deleted and screened for their impact on cell viability and titer in a FFA producing E. coli. Deletions of acrAB and rob, and to a lower degree of statistical confidence emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB(-) emrAB(-) strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB(+) producing strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate in an acrAB(-) strain. The findings suggest that AcrAB-TolC is responsible for the majority of FFA efflux in E. coli with residual activity provided by other RND-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts.
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