<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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2020-04-01
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| Series: | mSystems |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSystems.00736-19 |
| _version_ | 1818742251052859392 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-18T02:09:32Z |
| format | Article |
| id | doaj.art-bc54cb1a39f54e40a55a7c5e0ee0755c |
| institution | Directory Open Access Journal |
| issn | 2379-5077 |
| language | English |
| last_indexed | 2024-12-18T02:09:32Z |
| publishDate | 2020-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSystems |
| spelling | doaj.art-bc54cb1a39f54e40a55a7c5e0ee0755c2022-12-21T21:24:31ZengAmerican Society for MicrobiologymSystems2379-50772020-04-015210.1128/mSystems.00736-19<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 TracersTyler B. Jacobson0Travis K. Korosh1David M. Stevenson2Charles Foster3Costas Maranas4Daniel G. Olson5Lee R. Lynd6Daniel Amador-Noguez7Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USACenter for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USACenter for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USACenter for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USACenter for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USACenter for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USACenter for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USACenter for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mSystems.00736-19microbial metabolismmetabolic flux analysisMFAmass spectrometrybiofuelsGibbs free energy |
| spellingShingle | 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 mSystems microbial metabolism metabolic flux analysis MFA mass spectrometry biofuels Gibbs free energy |
| 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_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_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_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 |
| topic | microbial metabolism metabolic flux analysis MFA mass spectrometry biofuels Gibbs free energy |
| url | https://journals.asm.org/doi/10.1128/mSystems.00736-19 |
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