Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering eng...
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Autor*in:	Sander, Kyle [verfasserIn] Asano, Keiji G. Bhandari, Deepak Van Berkel, Gary J. Brown, Steven D. Davison, Brian Tschaplinski, Timothy J.

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2017

Schlagwörter:	Redox Adenylate energy charge Targeted metabolomics

Anmerkung:	© The Author(s) 2017

Übergeordnetes Werk:	Enthalten in: Biotechnology for biofuels - London : BioMed Central, 2008, 10(2017), 1 vom: 30. Nov.
Übergeordnetes Werk:	volume:10 ; year:2017 ; number:1 ; day:30 ; month:11

Links:	Volltext

DOI / URN:	10.1186/s13068-017-0960-4

Katalog-ID:	SPR030154537

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520			\|a Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types.
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allfields	10.1186/s13068-017-0960-4 doi (DE-627)SPR030154537 (SPR)s13068-017-0960-4-e DE-627 ger DE-627 rakwb eng Sander, Kyle verfasserin aut Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. Redox (dpeaa)DE-He213 Adenylate energy charge (dpeaa)DE-He213 Targeted metabolomics (dpeaa)DE-He213 Asano, Keiji G. aut Bhandari, Deepak aut Van Berkel, Gary J. aut Brown, Steven D. aut Davison, Brian aut Tschaplinski, Timothy J. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 10(2017), 1 vom: 30. Nov. (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:10 year:2017 number:1 day:30 month:11 https://dx.doi.org/10.1186/s13068-017-0960-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2119 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 30 11
spelling	10.1186/s13068-017-0960-4 doi (DE-627)SPR030154537 (SPR)s13068-017-0960-4-e DE-627 ger DE-627 rakwb eng Sander, Kyle verfasserin aut Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. Redox (dpeaa)DE-He213 Adenylate energy charge (dpeaa)DE-He213 Targeted metabolomics (dpeaa)DE-He213 Asano, Keiji G. aut Bhandari, Deepak aut Van Berkel, Gary J. aut Brown, Steven D. aut Davison, Brian aut Tschaplinski, Timothy J. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 10(2017), 1 vom: 30. Nov. (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:10 year:2017 number:1 day:30 month:11 https://dx.doi.org/10.1186/s13068-017-0960-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2119 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 30 11
allfields_unstemmed	10.1186/s13068-017-0960-4 doi (DE-627)SPR030154537 (SPR)s13068-017-0960-4-e DE-627 ger DE-627 rakwb eng Sander, Kyle verfasserin aut Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. Redox (dpeaa)DE-He213 Adenylate energy charge (dpeaa)DE-He213 Targeted metabolomics (dpeaa)DE-He213 Asano, Keiji G. aut Bhandari, Deepak aut Van Berkel, Gary J. aut Brown, Steven D. aut Davison, Brian aut Tschaplinski, Timothy J. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 10(2017), 1 vom: 30. Nov. (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:10 year:2017 number:1 day:30 month:11 https://dx.doi.org/10.1186/s13068-017-0960-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2119 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 30 11
allfieldsGer	10.1186/s13068-017-0960-4 doi (DE-627)SPR030154537 (SPR)s13068-017-0960-4-e DE-627 ger DE-627 rakwb eng Sander, Kyle verfasserin aut Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. Redox (dpeaa)DE-He213 Adenylate energy charge (dpeaa)DE-He213 Targeted metabolomics (dpeaa)DE-He213 Asano, Keiji G. aut Bhandari, Deepak aut Van Berkel, Gary J. aut Brown, Steven D. aut Davison, Brian aut Tschaplinski, Timothy J. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 10(2017), 1 vom: 30. Nov. (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:10 year:2017 number:1 day:30 month:11 https://dx.doi.org/10.1186/s13068-017-0960-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2119 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 30 11
allfieldsSound	10.1186/s13068-017-0960-4 doi (DE-627)SPR030154537 (SPR)s13068-017-0960-4-e DE-627 ger DE-627 rakwb eng Sander, Kyle verfasserin aut Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2017 Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. Redox (dpeaa)DE-He213 Adenylate energy charge (dpeaa)DE-He213 Targeted metabolomics (dpeaa)DE-He213 Asano, Keiji G. aut Bhandari, Deepak aut Van Berkel, Gary J. aut Brown, Steven D. aut Davison, Brian aut Tschaplinski, Timothy J. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 10(2017), 1 vom: 30. Nov. (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:10 year:2017 number:1 day:30 month:11 https://dx.doi.org/10.1186/s13068-017-0960-4 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2027 GBV_ILN_2055 GBV_ILN_2108 GBV_ILN_2111 GBV_ILN_2119 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 30 11
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author	Sander, Kyle
spellingShingle	Sander, Kyle misc Redox misc Adenylate energy charge misc Targeted metabolomics Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum
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topic_title	Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Redox (dpeaa)DE-He213 Adenylate energy charge (dpeaa)DE-He213 Targeted metabolomics (dpeaa)DE-He213
topic	misc Redox misc Adenylate energy charge misc Targeted metabolomics
topic_unstemmed	misc Redox misc Adenylate energy charge misc Targeted metabolomics
topic_browse	misc Redox misc Adenylate energy charge misc Targeted metabolomics
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title	Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum
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title_full	Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum
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author_browse	Sander, Kyle Asano, Keiji G. Bhandari, Deepak Van Berkel, Gary J. Brown, Steven D. Davison, Brian Tschaplinski, Timothy J.
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title_sort	targeted redox and energy cofactor metabolomics in clostridium thermocellum and thermoanaerobacterium saccharolyticum
title_auth	Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum
abstract	Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. © The Author(s) 2017
abstractGer	Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. © The Author(s) 2017
abstract_unstemmed	Background Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types. © The Author(s) 2017
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title_short	Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum
url	https://dx.doi.org/10.1186/s13068-017-0960-4
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author2	Asano, Keiji G. Bhandari, Deepak Van Berkel, Gary J. Brown, Steven D. Davison, Brian Tschaplinski, Timothy J.
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up_date	2024-07-03T14:18:59.360Z
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The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. 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Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

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