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Title: Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance

Abstract

Background: Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP. Results: Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an aldehyde dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrations found in acid-pretreated biomass. The mechanism of detoxification of HMF was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme. Conclusions: Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Expression of genes that reduce the effectmore » of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.« less

Authors:
 [1];  [1];  [2];  [3];  [1]
+ Show Author Affiliations
  1. Univ. of Georgia, Athens, GA (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1350020
Alternate Identifier(s):
OSTI ID: 1361373
Report Number(s):
NREL/JA-2700-68259
Journal ID: ISSN 1754-6834
Grant/Contract Number:  
AC36-08GO28308; AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; consolidated bioprocessing; Clostridium thermocellum; butanol dehydrogenase; furfural; 5-hydroxymethyl-2-furfural

Citation Formats

Kim, Sun -Ki, Groom, Joseph, Chung, Daehwan, Elkins, James, and Westpheling, Janet. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance. United States: N. p., 2017. Web. doi:10.1186/s13068-017-0750-z.
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Kim, Sun -Ki, Groom, Joseph, Chung, Daehwan, Elkins, James, & Westpheling, Janet. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance. United States. https://doi.org/10.1186/s13068-017-0750-z
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Kim, Sun -Ki, Groom, Joseph, Chung, Daehwan, Elkins, James, and Westpheling, Janet. Wed . "Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance". United States. https://doi.org/10.1186/s13068-017-0750-z. https://www.osti.gov/servlets/purl/1350020.
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@article{osti_1350020,
title = {Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance},
author = {Kim, Sun -Ki and Groom, Joseph and Chung, Daehwan and Elkins, James and Westpheling, Janet},
abstractNote = {Background: Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP. Results: Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an aldehyde dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrations found in acid-pretreated biomass. The mechanism of detoxification of HMF was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme. Conclusions: Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Expression of genes that reduce the effect of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.},
doi = {10.1186/s13068-017-0750-z},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 10,
place = {United States},
year = {2017},
month = {3}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1186/s13068-017-0750-z
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Figures / Tables:

Fig. 1 Fig. 1: Maps of shuttle vectors for BdhA expression in $C. thermocellum$. The bdhA gene from $T. pseudethanolicus$ 39E (Teth39_1597) was expressed under the control of the S‑layer (a), Clo1313_1809 (b), and enolase (c) promoters. Shuttle vectors contain a C‑terminal 6X His‑tag version of bdhA, a Rho‑independent terminator, the pyrFmore » (from $C. bescii$) cassette for selection of plasmid transformants, and pBAS2 sequences for replication in $C. therrmocellum$« less
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    Firmicutes-enriched IS1447 represents a group of IS3-family insertion sequences exhibiting unique + 1 transcriptional slippage
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