A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism
Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irr...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Barstow, Buz [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2011 |
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| Anmerkung: |
© Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( |
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| Übergeordnetes Werk: |
Enthalten in: Journal of biological engineering - Berlin : Springer, 2007, 5(2011), 1 vom: 26. Mai |
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| Übergeordnetes Werk: |
volume:5 ; year:2011 ; number:1 ; day:26 ; month:05 |
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| DOI / URN: |
10.1186/1754-1611-5-7 |
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| Katalog-ID: |
SPR029567149 |
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| 245 | 1 | 2 | |a A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism |
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| 520 | |a Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. | ||
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| 650 | 4 | |a Clostridium Acetobutylicum |7 (dpeaa)DE-He213 | |
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| 650 | 4 | |a Maturation Factor |7 (dpeaa)DE-He213 | |
| 700 | 1 | |a Agapakis, Christina M |4 aut | |
| 700 | 1 | |a Boyle, Patrick M |4 aut | |
| 700 | 1 | |a Grandl, Gerald |4 aut | |
| 700 | 1 | |a Silver, Pamela A |4 aut | |
| 700 | 1 | |a Wintermute, Edwin H |4 aut | |
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10.1186/1754-1611-5-7 doi (DE-627)SPR029567149 (SPR)1754-1611-5-7-e DE-627 ger DE-627 rakwb eng Barstow, Buz verfasserin aut A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. Genetic Selection (dpeaa)DE-He213 Clostridium Acetobutylicum (dpeaa)DE-He213 Hydrogenase Activity (dpeaa)DE-He213 Sulfite Reductase (dpeaa)DE-He213 Maturation Factor (dpeaa)DE-He213 Agapakis, Christina M aut Boyle, Patrick M aut Grandl, Gerald aut Silver, Pamela A aut Wintermute, Edwin H aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 5(2011), 1 vom: 26. Mai (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:5 year:2011 number:1 day:26 month:05 https://dx.doi.org/10.1186/1754-1611-5-7 lizenzpflichtig 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_39 GBV_ILN_40 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_2111 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_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2011 1 26 05 |
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10.1186/1754-1611-5-7 doi (DE-627)SPR029567149 (SPR)1754-1611-5-7-e DE-627 ger DE-627 rakwb eng Barstow, Buz verfasserin aut A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. Genetic Selection (dpeaa)DE-He213 Clostridium Acetobutylicum (dpeaa)DE-He213 Hydrogenase Activity (dpeaa)DE-He213 Sulfite Reductase (dpeaa)DE-He213 Maturation Factor (dpeaa)DE-He213 Agapakis, Christina M aut Boyle, Patrick M aut Grandl, Gerald aut Silver, Pamela A aut Wintermute, Edwin H aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 5(2011), 1 vom: 26. Mai (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:5 year:2011 number:1 day:26 month:05 https://dx.doi.org/10.1186/1754-1611-5-7 lizenzpflichtig 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_39 GBV_ILN_40 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_2111 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_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2011 1 26 05 |
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10.1186/1754-1611-5-7 doi (DE-627)SPR029567149 (SPR)1754-1611-5-7-e DE-627 ger DE-627 rakwb eng Barstow, Buz verfasserin aut A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. Genetic Selection (dpeaa)DE-He213 Clostridium Acetobutylicum (dpeaa)DE-He213 Hydrogenase Activity (dpeaa)DE-He213 Sulfite Reductase (dpeaa)DE-He213 Maturation Factor (dpeaa)DE-He213 Agapakis, Christina M aut Boyle, Patrick M aut Grandl, Gerald aut Silver, Pamela A aut Wintermute, Edwin H aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 5(2011), 1 vom: 26. Mai (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:5 year:2011 number:1 day:26 month:05 https://dx.doi.org/10.1186/1754-1611-5-7 lizenzpflichtig 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_39 GBV_ILN_40 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_2111 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_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2011 1 26 05 |
| allfieldsGer |
10.1186/1754-1611-5-7 doi (DE-627)SPR029567149 (SPR)1754-1611-5-7-e DE-627 ger DE-627 rakwb eng Barstow, Buz verfasserin aut A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. Genetic Selection (dpeaa)DE-He213 Clostridium Acetobutylicum (dpeaa)DE-He213 Hydrogenase Activity (dpeaa)DE-He213 Sulfite Reductase (dpeaa)DE-He213 Maturation Factor (dpeaa)DE-He213 Agapakis, Christina M aut Boyle, Patrick M aut Grandl, Gerald aut Silver, Pamela A aut Wintermute, Edwin H aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 5(2011), 1 vom: 26. Mai (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:5 year:2011 number:1 day:26 month:05 https://dx.doi.org/10.1186/1754-1611-5-7 lizenzpflichtig 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_39 GBV_ILN_40 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_2111 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_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2011 1 26 05 |
| allfieldsSound |
10.1186/1754-1611-5-7 doi (DE-627)SPR029567149 (SPR)1754-1611-5-7-e DE-627 ger DE-627 rakwb eng Barstow, Buz verfasserin aut A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. Genetic Selection (dpeaa)DE-He213 Clostridium Acetobutylicum (dpeaa)DE-He213 Hydrogenase Activity (dpeaa)DE-He213 Sulfite Reductase (dpeaa)DE-He213 Maturation Factor (dpeaa)DE-He213 Agapakis, Christina M aut Boyle, Patrick M aut Grandl, Gerald aut Silver, Pamela A aut Wintermute, Edwin H aut Enthalten in Journal of biological engineering Berlin : Springer, 2007 5(2011), 1 vom: 26. Mai (DE-627)54689884X (DE-600)2391582-1 1754-1611 nnns volume:5 year:2011 number:1 day:26 month:05 https://dx.doi.org/10.1186/1754-1611-5-7 lizenzpflichtig 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_39 GBV_ILN_40 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_2111 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_4338 GBV_ILN_4367 GBV_ILN_4700 AR 5 2011 1 26 05 |
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. 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A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism Genetic Selection (dpeaa)DE-He213 Clostridium Acetobutylicum (dpeaa)DE-He213 Hydrogenase Activity (dpeaa)DE-He213 Sulfite Reductase (dpeaa)DE-He213 Maturation Factor (dpeaa)DE-He213 |
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synthetic system links fefe-hydrogenases to essential e. coli sulfur metabolism |
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A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism |
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Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( |
| abstractGer |
Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( |
| abstract_unstemmed |
Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts. © Barstow et al.; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License ( |
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background FeFe-hydrogenases are the most active class of $ H_{2} $-producing enzymes known in nature and may have important applications in clean $ H_{2} $ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by $ O_{2} $. Results We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate $ O_{2} $ levels eliminate growth. This pathway forms the basis for a genetic selection for $ O_{2} $ tolerance. Genetically selected hydrogenases did not show improved stability in $ O_{2} $ and in many cases had lost $ H_{2} $ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. Conclusions Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic $ H_{2} $ metabolism. Our results also indicate a $ H_{2} $-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of $ H_{2} $-activating catalysts.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Genetic Selection</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Clostridium Acetobutylicum</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Hydrogenase Activity</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Sulfite Reductase</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Maturation Factor</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Agapakis, Christina M</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Boyle, Patrick M</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Grandl, Gerald</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Silver, Pamela A</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Wintermute, Edwin H</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">Journal of biological engineering</subfield><subfield code="d">Berlin : Springer, 2007</subfield><subfield code="g">5(2011), 1 vom: 26. 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