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Title: Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production

Abstract

Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains.

Authors:
ORCiD logo [1];  [1];  [2];  [3];  [2];  [1];  [1];  [3];  [1]
  1. Dartmouth College, Hanover, NH (United States). Thayer School of Engineering; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation
  2. Dartmouth College, Hanover, NH (United States). Thayer School of Engineering
  3. Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1627007
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 13; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
Biotechnology & Applied Microbiology; Energy & Fuels

Citation Formats

Holwerda, Evert K., Olson, Daniel G., Ruppertsberger, Natalie M., Stevenson, David M., Murphy, Sean J. L., Maloney, Marybeth I., Lanahan, Anthony A., Amador-Noguez, Daniel, and Lynd, Lee R. Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production. United States: N. p., 2020. Web. doi:10.1186/s13068-020-01680-5.
Holwerda, Evert K., Olson, Daniel G., Ruppertsberger, Natalie M., Stevenson, David M., Murphy, Sean J. L., Maloney, Marybeth I., Lanahan, Anthony A., Amador-Noguez, Daniel, & Lynd, Lee R. Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production. United States. https://doi.org/10.1186/s13068-020-01680-5
Holwerda, Evert K., Olson, Daniel G., Ruppertsberger, Natalie M., Stevenson, David M., Murphy, Sean J. L., Maloney, Marybeth I., Lanahan, Anthony A., Amador-Noguez, Daniel, and Lynd, Lee R. Tue . "Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production". United States. https://doi.org/10.1186/s13068-020-01680-5. https://www.osti.gov/servlets/purl/1627007.
@article{osti_1627007,
title = {Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production},
author = {Holwerda, Evert K. and Olson, Daniel G. and Ruppertsberger, Natalie M. and Stevenson, David M. and Murphy, Sean J. L. and Maloney, Marybeth I. and Lanahan, Anthony A. and Amador-Noguez, Daniel and Lynd, Lee R.},
abstractNote = {Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains.},
doi = {10.1186/s13068-020-01680-5},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 13,
place = {United States},
year = {Tue Mar 10 00:00:00 EDT 2020},
month = {Tue Mar 10 00:00:00 EDT 2020}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Figures / Tables:

Fig. 1 Fig. 1: Overview of the strain lineages and data associated with each strain (a) and overview of the research approach and data analysis in this paper (b). A total of 16 strains were analyzed, covering three different lineages. For each stage of the analysis different data sets were collected. Fourmore » strains (LL1011, LL1043, LL374 and LL375) have been subjected to Adaptive Laboratory Evolution, as indicated in the figure. For description of the strain, the adaptation, data generation and analysis see “Methods” section« less

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A defined growth medium with very low background carbon for culturing Clostridium thermocellum
journal, February 2012

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Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways
journal, May 2013

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  • Journal of Industrial Microbiology & Biotechnology, Vol. 40, Issue 7
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Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405)
journal, January 2012


Production of amino acids – Genetic and metabolic engineering approaches
journal, December 2017


Engineering Cellular Metabolism
journal, March 2016


Bio-based production of chemicals, materials and fuels – Corynebacterium glutamicum as versatile cell factory
journal, August 2012


Ethanol production by engineered thermophiles
journal, June 2015


Process engineering of cellulosic n-butanol production from corn-based biomass using Clostridium cellulovorans
journal, November 2017


From zero to hero—Design-based systems metabolic engineering of Corynebacterium glutamicum for l-lysine production
journal, March 2011


Redirecting carbon flux through exogenous pyruvate kinase to achieve high ethanol yields in Clostridium thermocellum
journal, January 2013


Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum
journal, September 2015


Metabolic and process engineering of Clostridium cellulovorans for biofuel production from cellulose
journal, November 2015


Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum
journal, November 2015


Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum
journal, November 2015


Fungal cellulases and complexed cellulosomal enzymes exhibit synergistic mechanisms in cellulose deconstruction
journal, January 2013

  • Resch, Michael G.; Donohoe, Bryon S.; Baker, John O.
  • Energy & Environmental Science, Vol. 6, Issue 6
  • DOI: 10.1039/c3ee00019b

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield
journal, September 2008

  • Shaw, A. J.; Podkaminer, K. K.; Desai, S. G.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 37, p. 13769-13774
  • DOI: 10.1073/pnas.0801266105

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum
journal, August 2011

  • Brown, S. D.; Guss, A. M.; Karpinets, T. V.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 33
  • DOI: 10.1073/pnas.1102444108

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii
journal, June 2014

  • Chung, D.; Cha, M.; Guss, A. M.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 24, p. 8931-8936
  • DOI: 10.1073/pnas.1402210111

Mutator action by Escherichia coli strains carrying dnaE mutations
journal, September 1977

  • Sevastopoulos, C. G.; Glaser, D. A.
  • Proceedings of the National Academy of Sciences, Vol. 74, Issue 9
  • DOI: 10.1073/pnas.74.9.3947

Stability of Glutamine and Pyroglutamic Acid under Model System Conditions: Influence of Physical and Technological Factors
journal, November 1987


Effects of Stirring and Hydrogen on Fermentation Products of Clostridium thermocellum
journal, January 1988


Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora
journal, January 2016


Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000
journal, January 2017


The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading
journal, October 2014

  • Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.
  • Biotechnology for Biofuels, Vol. 7, Issue 1
  • DOI: 10.1186/s13068-014-0155-1

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

  • Biswas, Ranjita; Zheng, Tianyong; Olson, Daniel G.
  • Biotechnology for Biofuels, Vol. 8, Issue 1
  • DOI: 10.1186/s13068-015-0204-4

Anaerobic detoxification of acetic acid in a thermophilic ethanologen
journal, May 2015

  • Shaw, A. Joe; Miller, Bethany B.; Rogers, Stephen R.
  • Biotechnology for Biofuels, Vol. 8, Issue 1
  • DOI: 10.1186/s13068-015-0257-4

Biological lignocellulose solubilization: comparative evaluation of biocatalysts and enhancement via cotreatment
journal, January 2016

  • Paye, Julie M. D.; Guseva, Anna; Hammer, Sarah K.
  • Biotechnology for Biofuels, Vol. 9, Issue 1
  • DOI: 10.1186/s13068-015-0412-y

Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood
journal, June 2016

  • Herring, Christopher D.; Kenealy, William R.; Joe Shaw, A.
  • Biotechnology for Biofuels, Vol. 9, Issue 1
  • DOI: 10.1186/s13068-016-0536-8

Deletion of the hfsB gene increases ethanol production in Thermoanaerobacterium saccharolyticum and several other thermophilic anaerobic bacteria
journal, November 2017

  • Eminoğlu, Ayşenur; Murphy, Sean Jean-Loup; Maloney, Marybeth
  • Biotechnology for Biofuels, Vol. 10, Issue 1
  • DOI: 10.1186/s13068-017-0968-9

Multiple levers for overcoming the recalcitrance of lignocellulosic biomass
journal, January 2019

  • Holwerda, Evert K.; Worthen, Robert S.; Kothari, Ninad
  • Biotechnology for Biofuels, Vol. 12, Issue 1
  • DOI: 10.1186/s13068-019-1353-7

Engineering Corynebacterium glutamicum triggers glutamic acid accumulation in biotin-rich corn stover hydrolysate
journal, April 2019


Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.