Development of a native Escherichia coli induction system for ionic liquid tolerance

PLoS One. 2014 Jul 1;9(7):e101115. doi: 10.1371/journal.pone.0101115. eCollection 2014.

Abstract

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Escherichia coli / drug effects*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Ionic Liquids / pharmacology*
  • Promoter Regions, Genetic
  • RNA, Bacterial / genetics
  • Transcriptome

Substances

  • EilA protein, E coli
  • Escherichia coli Proteins
  • Ionic Liquids
  • RNA, Bacterial

Grants and funding

This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. Kimmo Hutter was supported by his Master program at the Mannheim University of Applied Sciences and Prof. Matthias Mack. William Turner and Mary Dunlop were supported by the Office of Science (BER) at the U.S. Department of Energy and the NASA Vermont Space Grant Consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.