title: Development of a native Escherichia coli induction system for ionic liquid tolerance.
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.
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Marijke
authors.initials: M
authors.lastname: Frederix
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Kimmo
authors.initials: K
authors.lastname: Hütter
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Jessica
authors.initials: J
authors.lastname: Leu
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Tanveer S
authors.initials: TS
authors.lastname: Batth
authors.affiliation: School of Engineering, University of Vermont, Burlington, Vermont, United States of America.
authors.forename: William J
authors.initials: WJ
authors.lastname: Turner
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Botanical Institute, University of Basel, Basel, Switzerland; Biology and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, United States of America.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Rüegg
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America; Department of Chemistry, College of Chemistry, University of California, Berkeley, California, United States of America.
authors.forename: Harvey W
authors.initials: HW
authors.lastname: Blanch
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Biological and Materials Science Center, Sandia National Laboratories, Livermore, California, United States of America.
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America; Department of Chemistry, College of Chemistry, University of California, Berkeley, California, United States of America; Department of Chemical & Biomolecular Engineering, College of Chemistry, University of California, Berkeley, California, United States of America.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Biology and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, United States of America.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Thelen
authors.affiliation: School of Engineering, University of Vermont, Burlington, Vermont, United States of America.
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Dunlop
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
mesh_terms: D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D052578:Ionic Liquids / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D059467:Transcriptome
chemical_list: C513921:EilA protein, E coli; D029968:Escherichia coli Proteins; D052578:Ionic Liquids; D012329:RNA, Bacterial