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Title: Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum

Abstract

Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, we study a mutant strain of C. thermocellum that was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulose Avicel, effectively eliminating formate, acetate, and lactate production and reducing H2 production by fivefold. On 5 g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4 mM in mutant AG553 on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growthmore » of C. thermocellum, likely due to CO2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1 mM ethanol on 10 g/L Avicel. In conclusion, with the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.« less

Authors:
 [1];  [2];  [2];  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1286864
Alternate Identifier(s):
OSTI ID: 1250941
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 32; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; Clostridium thermocellum; biofuels; metabolic engineering

Citation Formats

Papanek, Beth A., Biswas, Ranjita, Rydzak, Thomas, and Guss, Adam M. Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum. United States: N. p., 2015. Web. doi:10.1016/j.ymben.2015.09.002.
Papanek, Beth A., Biswas, Ranjita, Rydzak, Thomas, & Guss, Adam M. Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum. United States. https://doi.org/10.1016/j.ymben.2015.09.002
Papanek, Beth A., Biswas, Ranjita, Rydzak, Thomas, and Guss, Adam M. 2015. "Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum". United States. https://doi.org/10.1016/j.ymben.2015.09.002. https://www.osti.gov/servlets/purl/1286864.
@article{osti_1286864,
title = {Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum},
author = {Papanek, Beth A. and Biswas, Ranjita and Rydzak, Thomas and Guss, Adam M.},
abstractNote = {Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, we study a mutant strain of C. thermocellum that was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulose Avicel, effectively eliminating formate, acetate, and lactate production and reducing H2 production by fivefold. On 5 g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4 mM in mutant AG553 on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to CO2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1 mM ethanol on 10 g/L Avicel. In conclusion, with the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.},
doi = {10.1016/j.ymben.2015.09.002},
url = {https://www.osti.gov/biblio/1286864}, journal = {Metabolic Engineering},
issn = {1096-7176},
number = ,
volume = 32,
place = {United States},
year = {Sat Sep 12 00:00:00 EDT 2015},
month = {Sat Sep 12 00:00:00 EDT 2015}
}

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Cited by: 59 works
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Works referenced in this record:

High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes
journal, September 2011


Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum
journal, January 2015


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journal, July 2015


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journal, January 2003


Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant
journal, August 2010


Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass
journal, January 2013


Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain
journal, January 2014


Works referencing / citing this record:

Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides
journal, December 2016


Consolidated bioprocessing of Populus using Clostridium (Ruminiclostridium) thermocellum: a case study on the impact of lignin composition and structure
journal, February 2016


Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum
journal, June 2016


Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase
journal, October 2017


Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics
journal, April 2018


Engineering Clostridium for improved solvent production: recent progress and perspective
journal, May 2019


Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol
journal, November 2017


Engineering microbes for direct fermentation of cellulose to bioethanol
journal, February 2018


Expression of adhA from different organisms in Clostridium thermocellum
journal, November 2017


Fuelling the future: microbial engineering for the production of sustainable biofuels
journal, March 2016


CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313
journal, August 2019


Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production : Evolutionary Engineering of
journal, April 2016


Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production
journal, March 2020


Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism
journal, January 2017


Rational development of transformation in Clostridium thermocellum ATCC 27405 via complete methylome analysis and evasion of native restriction–modification systems
journal, July 2019


Nicotinamide cofactor ratios in engineered strains of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum
journal, April 2016


Agronomic performance of Populus deltoides trees engineered for biofuel production
journal, November 2017


Consolidated bioprocessing of Populus using Clostridium (Ruminiclostridium) thermocellum: a case study on the impact of lignin composition and structure
journal, February 2016


Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism
journal, January 2017


Agronomic performance of Populus deltoides trees engineered for biofuel production
journal, November 2017


Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol
journal, November 2017


Progress in understanding and overcoming biomass recalcitrance: a BioEnergy Science Center (BESC) perspective
journal, November 2017


Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics
journal, April 2018


Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production
journal, March 2020