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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

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. Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrations found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural 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. Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Lastly, expression of genes that reduce the effect of thesemore » 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]
  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) (SC-23)
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:
Journal Article: 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.
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. doi:10.1186/s13068-017-0750-z.
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. doi:10.1186/s13068-017-0750-z. https://www.osti.gov/servlets/purl/1350020.
@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 = {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. Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrations found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural 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. Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Lastly, 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 = {Wed Mar 15 00:00:00 EDT 2017},
month = {Wed Mar 15 00:00:00 EDT 2017}
}

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  • 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 derivativesmore » 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.« less
  • Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less
  • An alcohol dehydrogenase reported from Thermoanaerobium brockii catalyzes the asymmetric reduction of aliphatic acyclic ketones. An interesting substrate size-induced reversal of stereoselectivity at 37/degree/C was observed by Keinan and co-workers in their studies of this latter enzyme. The smaller substrates (2-butanone, 3-methyl-2-butanone, or methyl cyclopropyl ketone) were reduced to (R)-alcohols, whereas 2-pentanone and the longer chain ketones provided the (S)-alcohols. In order to evaluate the mechanistic basis of this unusual stereochemical reversal, the authors have studied the kinetics of the reaction of SADH from T. ethanolicus with the (R)- and (S)-enantiomers of the simple secondary alcohols, 2-butanol and 2-pentanol.
  • Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. As a result, towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.