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Title: In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers

Abstract

ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with 2 H and 13 C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli . The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellum than in T. saccharolyticum . These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA)more » cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. IMPORTANCE Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum , two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such as E. coli or Saccharomyces cerevisiae , the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1604827
Grant/Contract Number:  
[AC05-00OR22725; SC0018998]
Resource Type:
Published Article
Journal Name:
mSystems
Additional Journal Information:
[Journal Name: mSystems Journal Volume: 5 Journal Issue: 2]; Journal ID: ISSN 2379-5077
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English

Citation Formats

Jacobson, Tyler B., Korosh, Travis K., Stevenson, David M., Foster, Charles, Maranas, Costas, Olson, Daniel G., Lynd, Lee R., Amador-Noguez, Daniel, and Porto, ed., Carla. In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers. United States: N. p., 2020. Web. doi:10.1128/mSystems.00736-19.
Jacobson, Tyler B., Korosh, Travis K., Stevenson, David M., Foster, Charles, Maranas, Costas, Olson, Daniel G., Lynd, Lee R., Amador-Noguez, Daniel, & Porto, ed., Carla. In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers. United States. doi:10.1128/mSystems.00736-19.
Jacobson, Tyler B., Korosh, Travis K., Stevenson, David M., Foster, Charles, Maranas, Costas, Olson, Daniel G., Lynd, Lee R., Amador-Noguez, Daniel, and Porto, ed., Carla. Tue . "In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers". United States. doi:10.1128/mSystems.00736-19.
@article{osti_1604827,
title = {In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers},
author = {Jacobson, Tyler B. and Korosh, Travis K. and Stevenson, David M. and Foster, Charles and Maranas, Costas and Olson, Daniel G. and Lynd, Lee R. and Amador-Noguez, Daniel and Porto, ed., Carla},
abstractNote = {ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with 2 H and 13 C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli . The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellum than in T. saccharolyticum . These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. IMPORTANCE Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum , two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such as E. coli or Saccharomyces cerevisiae , the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.},
doi = {10.1128/mSystems.00736-19},
journal = {mSystems},
number = [2],
volume = [5],
place = {United States},
year = {2020},
month = {3}
}

Journal Article:
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DOI: 10.1128/mSystems.00736-19

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