title: Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol.
abstract: The microbial conversion of sugars to fuels is a promising technology, but 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 engineer efflux pumps to secrete fuels and inhibitory chemicals from the cell, increasing microbial tolerance and enabling higher fuel titer. Toward that end, we used a directed evolution strategy to generate variants of the Escherichia coli AcrB efflux pump that act on the non-native substrate n-butanol, enhancing growth rates of E. coli in the presence of this biofuel by up to 25%. Furthermore, these variants confer improved tolerance to isobutanol and straight-chain alcohols up to n-heptanol. Single amino acid changes in AcrB responsible for this phenotype were identified. We have also shown that both the chemical and genetic inactivation of pump activity eliminate the tolerance conferred by AcrB pump variants, supporting our assertion that the variants secrete the non-native substrates. This strategy can be applied to create an array of efflux pumps that modulate the intracellular concentrations of small molecules of interest to microbial fuel and chemical production.
authors.affiliation: Energy Biosciences Institute, University of California Berkeley , 2151 Berkeley Way, Berkeley, California 94704, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Fisher
authors.forename: Sergey
authors.initials: S
authors.lastname: Boyarskiy
authors.forename: Masaki R
authors.initials: MR
authors.lastname: Yamada
authors.forename: Niwen
authors.initials: N
authors.lastname: Kong
authors.forename: Stefan
authors.initials: S
authors.lastname: Bauer
authors.forename: Danielle
authors.initials: D
authors.lastname: Tullman-Ercek
mesh_terms: D020001:1-Butanol / Q000737:chemistry / Q000378:metabolism*; D056804:Biofuels; D000440:Butanols / Q000737:chemistry / Q000378:metabolism; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D020001:1-Butanol; D056804:Biofuels; D000440:Butanols; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli; C040507:isobutyl alcohol