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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 majormore » 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.« less

Authors:
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Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1353358
Report Number(s):
PNNL-SA-106710
Journal ID: ISSN 1664-302X; 47418; KP1601050
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Frontiers in Microbiology; Journal Volume: 5
Country of Publication:
United States
Language:
English
Subject:
aromatic inhibitors; ammonia-pretreated lignocellulose; ethanologenesis; detoxification; Environmental Molecular Sciences Laboratory

Citation Formats

Keating, David H., Zhang, Yaoping, Ong, Irene M., McIlwain, Sean, Morales, Eduardo H., Grass, Jeffrey A., Tremaine, Mary, Bothfeld, William, Higbee, Alan, Ulbrich, Arne, Balloon, Allison J., Westphall, Michael S., Aldrich, Josh, Lipton, Mary S., Kim, Joonhoon, Moskvin, Oleg V., Bukhman, Yury V., Coon, Joshua J., Kiley, Patricia J., Bates, Donna M., and Landick, Robert. Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification. United States: N. p., 2014. Web. doi:10.3389/fmicb.2014.00402.
Keating, David H., Zhang, Yaoping, Ong, Irene M., McIlwain, Sean, Morales, Eduardo H., Grass, Jeffrey A., Tremaine, Mary, Bothfeld, William, Higbee, Alan, Ulbrich, Arne, Balloon, Allison J., Westphall, Michael S., Aldrich, Josh, Lipton, Mary S., Kim, Joonhoon, Moskvin, Oleg V., Bukhman, Yury V., Coon, Joshua J., Kiley, Patricia J., Bates, Donna M., & Landick, Robert. Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification. United States. doi:10.3389/fmicb.2014.00402.
Keating, David H., Zhang, Yaoping, Ong, Irene M., McIlwain, Sean, Morales, Eduardo H., Grass, Jeffrey A., Tremaine, Mary, Bothfeld, William, Higbee, Alan, Ulbrich, Arne, Balloon, Allison J., Westphall, Michael S., Aldrich, Josh, Lipton, Mary S., Kim, Joonhoon, Moskvin, Oleg V., Bukhman, Yury V., Coon, Joshua J., Kiley, Patricia J., Bates, Donna M., and Landick, Robert. Wed . "Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification". United States. doi:10.3389/fmicb.2014.00402.
@article{osti_1353358,
title = {Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification},
author = {Keating, David H. and Zhang, Yaoping and Ong, Irene M. and McIlwain, Sean and Morales, Eduardo H. and Grass, Jeffrey A. and Tremaine, Mary and Bothfeld, William and Higbee, Alan and Ulbrich, Arne and Balloon, Allison J. and Westphall, Michael S. and Aldrich, Josh and Lipton, Mary S. and Kim, Joonhoon and Moskvin, Oleg V. and Bukhman, Yury V. and Coon, Joshua J. and Kiley, Patricia J. and Bates, Donna M. and Landick, Robert},
abstractNote = {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.},
doi = {10.3389/fmicb.2014.00402},
journal = {Frontiers in Microbiology},
number = ,
volume = 5,
place = {United States},
year = {Wed Aug 13 00:00:00 EDT 2014},
month = {Wed Aug 13 00:00:00 EDT 2014}
}