<italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers

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 2H and...

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Autores principales:	Tyler B. Jacobson, Travis K. Korosh, David M. Stevenson, Charles Foster, Costas Maranas, Daniel G. Olson, Lee R. Lynd, Daniel Amador-Noguez
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	microbial metabolism metabolic flux analysis MFA mass spectrometry biofuels Gibbs free energy Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bc54cb1a39f54e40a55a7c5e0ee0755c
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spelling	oai:doaj.org-article:bc54cb1a39f54e40a55a7c5e0ee0755c2021-12-02T19:46:20Z<italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers10.1128/mSystems.00736-192379-5077https://doaj.org/article/bc54cb1a39f54e40a55a7c5e0ee0755c2020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00736-19https://doaj.org/toc/2379-5077ABSTRACT 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 2H and 13C 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.Tyler B. JacobsonTravis K. KoroshDavid M. StevensonCharles FosterCostas MaranasDaniel G. OlsonLee R. LyndDaniel Amador-NoguezAmerican Society for Microbiologyarticlemicrobial metabolismmetabolic flux analysisMFAmass spectrometrybiofuelsGibbs free energyMicrobiologyQR1-502ENmSystems, Vol 5, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	microbial metabolism metabolic flux analysis MFA mass spectrometry biofuels Gibbs free energy Microbiology QR1-502
spellingShingle	microbial metabolism metabolic flux analysis MFA mass spectrometry biofuels Gibbs free energy Microbiology QR1-502 Tyler B. Jacobson Travis K. Korosh David M. Stevenson Charles Foster Costas Maranas Daniel G. Olson Lee R. Lynd Daniel Amador-Noguez <italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers
description	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 2H and 13C 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.
format	article
author	Tyler B. Jacobson Travis K. Korosh David M. Stevenson Charles Foster Costas Maranas Daniel G. Olson Lee R. Lynd Daniel Amador-Noguez
author_facet	Tyler B. Jacobson Travis K. Korosh David M. Stevenson Charles Foster Costas Maranas Daniel G. Olson Lee R. Lynd Daniel Amador-Noguez
author_sort	Tyler B. Jacobson
title	<italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers
title_short	<italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers
title_full	<italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers
title_fullStr	<italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers
title_full_unstemmed	<italic toggle="yes">In Vivo</italic> Thermodynamic Analysis of Glycolysis in <named-content content-type="genus-species">Clostridium thermocellum</named-content> and <named-content content-type="genus-species">Thermoanaerobacterium saccharolyticum</named-content> Using <sup>13</sup>C and <sup>2</sup>H Tracers
title_sort	<italic toggle="yes">in vivo</italic> thermodynamic analysis of glycolysis in <named-content content-type="genus-species">clostridium thermocellum</named-content> and <named-content content-type="genus-species">thermoanaerobacterium saccharolyticum</named-content> using <sup>13</sup>c and <sup>2</sup>h tracers
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/bc54cb1a39f54e40a55a7c5e0ee0755c
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