title: Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification.
abstract: Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Keating
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Yaoping
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Irene M
authors.initials: IM
authors.lastname: Ong
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Sean
authors.initials: S
authors.lastname: McIlwain
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA ; Department of Biomolecular Chemistry, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Eduardo H
authors.initials: EH
authors.lastname: Morales
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA ; Department of Biochemistry, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Grass
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Mary
authors.initials: M
authors.lastname: Tremaine
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: William
authors.initials: W
authors.lastname: Bothfeld
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Higbee
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Arne
authors.initials: A
authors.lastname: Ulbrich
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Allison J
authors.initials: AJ
authors.lastname: Balloon
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin-Madison Madison, WI, USA ; Department of Chemistry, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.affiliation: Pacific Northwest National Laboratory Richland, WA, USA.
authors.forename: Josh
authors.initials: J
authors.lastname: Aldrich
authors.affiliation: Pacific Northwest National Laboratory Richland, WA, USA.
authors.forename: Mary S
authors.initials: MS
authors.lastname: Lipton
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA ; Department of Chemical and Biological Engineering, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Joonhoon
authors.initials: J
authors.lastname: Kim
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Moskvin
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Yury V
authors.initials: YV
authors.lastname: Bukhman
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA ; Department of Biomolecular Chemistry, University of Wisconsin-Madison Madison, WI, USA ; Department of Chemistry, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA ; Department of Biomolecular Chemistry, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Kiley
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Bates
authors.affiliation: Great Lakes Bioenergy Research Center, University of Wisconsin-Madison Madison, WI, USA ; Department of Biochemistry, University of Wisconsin-Madison Madison, WI, USA ; Department of Bacteriology, University of Wisconsin-Madison Madison, WI, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
keywords: Escherichia coli; RNAseq; aromatic inhibitors; biofuels; ethanol; lignocellulosic hydrolysate; proteomics; transcriptomics