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Title: A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

Abstract

Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold bymore » furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr- and isobutyraldehydes. Conclusions: Thermoanaerobacter pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomics analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.« less

Authors:
 [1];  [2];  [1];  [1];  [1];  [1];  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Desert Research Institute, Las Vegas, NV (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1185570
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; thermophiles; proteomics; inhibitor; biomass; pretreatment; furfural; 5-hydroxymethyl furfural; butanol dehydrogenase

Citation Formats

Clarkson, Sonya M., Hamilton-Brehm, Scott D., Giannone, Richard J., Engle, Nancy L., Tschaplinski, Timothy J., Hettich, Robert L., and Elkins, James G. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E. United States: N. p., 2014. Web. doi:10.1186/s13068-014-0165-z.
Clarkson, Sonya M., Hamilton-Brehm, Scott D., Giannone, Richard J., Engle, Nancy L., Tschaplinski, Timothy J., Hettich, Robert L., & Elkins, James G. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E. United States. doi:10.1186/s13068-014-0165-z.
Clarkson, Sonya M., Hamilton-Brehm, Scott D., Giannone, Richard J., Engle, Nancy L., Tschaplinski, Timothy J., Hettich, Robert L., and Elkins, James G. Wed . "A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E". United States. doi:10.1186/s13068-014-0165-z. https://www.osti.gov/servlets/purl/1185570.
@article{osti_1185570,
title = {A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E},
author = {Clarkson, Sonya M. and Hamilton-Brehm, Scott D. and Giannone, Richard J. and Engle, Nancy L. and Tschaplinski, Timothy J. and Hettich, Robert L. and Elkins, James G.},
abstractNote = {Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr- and isobutyraldehydes. Conclusions: Thermoanaerobacter pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomics analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.},
doi = {10.1186/s13068-014-0165-z},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 7,
place = {United States},
year = {Wed Dec 03 00:00:00 EST 2014},
month = {Wed Dec 03 00:00:00 EST 2014}
}

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