Enhancing tolerance to short-chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non-native substrate n-butanol

Michael A. Fisher, Sergey Boyarskiy, Masaki R. Yamada, Niwen Kong, Stefan Bauer, Danielle Tullman-Ercek*

*Corresponding author for this work

Research output: Contribution to journalArticle

59 Scopus citations

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 straightchain 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.

Original languageEnglish (US)
Pages (from-to)30-40
Number of pages11
JournalACS synthetic biology
Volume3
Issue number1
DOIs
StatePublished - Jan 17 2014

Keywords

  • Biofuels
  • Directed evolution
  • Efflux pump
  • Protein engineering
  • Tolerance
  • Transporter

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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