S96: Engineering efflux pumps to enhance microbial secretion of biofuel-relevant inhibitors

The microbial conversion of sugars to biofuels is a promising new technology, but the fuels are usually inefficiently recovered from the microbial production host. Furthermore, the byproducts of biomass pretreatment processes and the fuels themselves are often toxic at industrially-relevant levels. One promising solution to these problems is to evolve efflux pumps to secrete fuels and inhibitory chemicals from the cell, thus increasing both fuel titer and microbial tolerance. Using a selective growth competition method combined with several mutagenesis schemes, we have isolated variants of the Escherichia coli AcrB efflux pump that enhance the growth of E. coli in the presence of n-butanol by approximately 25%. The growth enhancement is maintained even as the concentration of inhibitor is increased. As each variant is comprised of several mutations, we have identified the single amino acid changes in AcrB that are responsible for the enhanced growth phenotype. Furthermore, we found that these variants enhance tolerance to isobutanol and straight-chain alcohols to n-heptanol, but not chloramphenicol. Moving forward, we will test if these pump variants enhance production of n-butanol and isobutanol from producing strains, carry out a second round of mutagenesis and selection, and screen our AcrB libraries against additional biofuel-relevant inhibitors.