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Clarkson SM, Hamilton-Brehm SD, Giannone RJ, et al. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E. Biotechnol Biofuels. 2014;7(1):165doi: 10.1186/s13068-014-0165-z.
Clarkson, S. M., Hamilton-Brehm, S. D., Giannone, R. J., Engle, N. L., Tschaplinski, T. J., Hettich, R. L., & Elkins, J. G. (2014). A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E. Biotechnology for biofuels, 7(1), 165. https://doi.org/10.1186/s13068-014-0165-z
Clarkson, Sonya M, et al. "A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E." Biotechnology for biofuels vol. 7,1 (2014): 165. doi: https://doi.org/10.1186/s13068-014-0165-z
Clarkson SM, Hamilton-Brehm SD, Giannone RJ, Engle NL, Tschaplinski TJ, Hettich RL, Elkins JG. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E. Biotechnol Biofuels. 2014 Dec 03;7(1):165. doi: 10.1186/s13068-014-0165-z. eCollection 2014. PMID: 25506391; PMCID: PMC4265447.
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Biotechnol Biofuels. 2014 Dec 03;7(1):165. doi: 10.1186/s13068-014-0165-z. eCollection 2014.

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

Biotechnology for biofuels

Sonya M Clarkson, Scott D Hamilton-Brehm, Richard J Giannone, Nancy L Engle, Timothy J Tschaplinski, Robert L Hettich, James G Elkins

Affiliations

  1. BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA.
  2. BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA ; Current address: Division of Earth and Ecosystem Sciences, Desert Research Institute, Las Vegas, NV USA.
  3. BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA ; Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6341 USA.

PMID: 25506391 PMCID: PMC4265447 DOI: 10.1186/s13068-014-0165-z

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 are 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: Thermoanaerobacter 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 versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold 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. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.

CONCLUSIONS: T. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic 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.

Keywords: 5-hydroxymethylfurfural; Biofuels; Butanol dehydrogenase; Furfural; Inhibitor; Lignocellulosic; Pretreatment; Proteomics; Thermophiles

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