Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose

Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferm...
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Autor*in:	Tian, Liang [verfasserIn] Conway, Peter M. Cervenka, Nicholas D. Cui, Jingxuan Maloney, Marybeth Olson, Daniel G. Lynd, Lee R.

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2019

Schlagwörter:	Cellulosic biofuel Consolidated bioprocessing -Butanol Protein engineering

Anmerkung:	© The Author(s) 2019

Übergeordnetes Werk:	Enthalten in: Biotechnology for biofuels - London : BioMed Central, 2008, 12(2019), 1 vom: 23. Juli
Übergeordnetes Werk:	volume:12 ; year:2019 ; number:1 ; day:23 ; month:07

Links:	Volltext

DOI / URN:	10.1186/s13068-019-1524-6

Katalog-ID:	SPR030160782

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520			\|a Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h.
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650		4	\|a -Butanol \|7 (dpeaa)DE-He213
650		4	\|a Protein engineering \|7 (dpeaa)DE-He213
700	1		\|a Conway, Peter M. \|4 aut
700	1		\|a Cervenka, Nicholas D. \|4 aut
700	1		\|a Cui, Jingxuan \|4 aut
700	1		\|a Maloney, Marybeth \|4 aut
700	1		\|a Olson, Daniel G. \|4 aut
700	1		\|a Lynd, Lee R. \|4 aut
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allfields	10.1186/s13068-019-1524-6 doi (DE-627)SPR030160782 (SPR)s13068-019-1524-6-e DE-627 ger DE-627 rakwb eng Tian, Liang verfasserin (orcid)0000-0002-6419-3695 aut Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2019 Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. Cellulosic biofuel (dpeaa)DE-He213 Consolidated bioprocessing (dpeaa)DE-He213 -Butanol (dpeaa)DE-He213 Protein engineering (dpeaa)DE-He213 Conway, Peter M. aut Cervenka, Nicholas D. aut Cui, Jingxuan aut Maloney, Marybeth aut Olson, Daniel G. aut Lynd, Lee R. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 12(2019), 1 vom: 23. Juli (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:12 year:2019 number:1 day:23 month:07 https://dx.doi.org/10.1186/s13068-019-1524-6 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 12 2019 1 23 07
spelling	10.1186/s13068-019-1524-6 doi (DE-627)SPR030160782 (SPR)s13068-019-1524-6-e DE-627 ger DE-627 rakwb eng Tian, Liang verfasserin (orcid)0000-0002-6419-3695 aut Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2019 Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. Cellulosic biofuel (dpeaa)DE-He213 Consolidated bioprocessing (dpeaa)DE-He213 -Butanol (dpeaa)DE-He213 Protein engineering (dpeaa)DE-He213 Conway, Peter M. aut Cervenka, Nicholas D. aut Cui, Jingxuan aut Maloney, Marybeth aut Olson, Daniel G. aut Lynd, Lee R. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 12(2019), 1 vom: 23. Juli (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:12 year:2019 number:1 day:23 month:07 https://dx.doi.org/10.1186/s13068-019-1524-6 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 12 2019 1 23 07
allfields_unstemmed	10.1186/s13068-019-1524-6 doi (DE-627)SPR030160782 (SPR)s13068-019-1524-6-e DE-627 ger DE-627 rakwb eng Tian, Liang verfasserin (orcid)0000-0002-6419-3695 aut Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2019 Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. Cellulosic biofuel (dpeaa)DE-He213 Consolidated bioprocessing (dpeaa)DE-He213 -Butanol (dpeaa)DE-He213 Protein engineering (dpeaa)DE-He213 Conway, Peter M. aut Cervenka, Nicholas D. aut Cui, Jingxuan aut Maloney, Marybeth aut Olson, Daniel G. aut Lynd, Lee R. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 12(2019), 1 vom: 23. Juli (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:12 year:2019 number:1 day:23 month:07 https://dx.doi.org/10.1186/s13068-019-1524-6 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 12 2019 1 23 07
allfieldsGer	10.1186/s13068-019-1524-6 doi (DE-627)SPR030160782 (SPR)s13068-019-1524-6-e DE-627 ger DE-627 rakwb eng Tian, Liang verfasserin (orcid)0000-0002-6419-3695 aut Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2019 Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. Cellulosic biofuel (dpeaa)DE-He213 Consolidated bioprocessing (dpeaa)DE-He213 -Butanol (dpeaa)DE-He213 Protein engineering (dpeaa)DE-He213 Conway, Peter M. aut Cervenka, Nicholas D. aut Cui, Jingxuan aut Maloney, Marybeth aut Olson, Daniel G. aut Lynd, Lee R. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 12(2019), 1 vom: 23. Juli (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:12 year:2019 number:1 day:23 month:07 https://dx.doi.org/10.1186/s13068-019-1524-6 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 12 2019 1 23 07
allfieldsSound	10.1186/s13068-019-1524-6 doi (DE-627)SPR030160782 (SPR)s13068-019-1524-6-e DE-627 ger DE-627 rakwb eng Tian, Liang verfasserin (orcid)0000-0002-6419-3695 aut Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2019 Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. Cellulosic biofuel (dpeaa)DE-He213 Consolidated bioprocessing (dpeaa)DE-He213 -Butanol (dpeaa)DE-He213 Protein engineering (dpeaa)DE-He213 Conway, Peter M. aut Cervenka, Nicholas D. aut Cui, Jingxuan aut Maloney, Marybeth aut Olson, Daniel G. aut Lynd, Lee R. aut Enthalten in Biotechnology for biofuels London : BioMed Central, 2008 12(2019), 1 vom: 23. Juli (DE-627)563167882 (DE-600)2421351-2 1754-6834 nnns volume:12 year:2019 number:1 day:23 month:07 https://dx.doi.org/10.1186/s13068-019-1524-6 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 12 2019 1 23 07
language	English
source	Enthalten in Biotechnology for biofuels 12(2019), 1 vom: 23. Juli volume:12 year:2019 number:1 day:23 month:07
sourceStr	Enthalten in Biotechnology for biofuels 12(2019), 1 vom: 23. Juli volume:12 year:2019 number:1 day:23 month:07
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Cellulosic biofuel Consolidated bioprocessing -Butanol Protein engineering
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container_title	Biotechnology for biofuels
authorswithroles_txt_mv	Tian, Liang @@aut@@ Conway, Peter M. @@aut@@ Cervenka, Nicholas D. @@aut@@ Cui, Jingxuan @@aut@@ Maloney, Marybeth @@aut@@ Olson, Daniel G. @@aut@@ Lynd, Lee R. @@aut@@
publishDateDaySort_date	2019-07-23T00:00:00Z
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topic_title	Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose Cellulosic biofuel (dpeaa)DE-He213 Consolidated bioprocessing (dpeaa)DE-He213 -Butanol (dpeaa)DE-He213 Protein engineering (dpeaa)DE-He213
topic	misc Cellulosic biofuel misc Consolidated bioprocessing misc -Butanol misc Protein engineering
topic_unstemmed	misc Cellulosic biofuel misc Consolidated bioprocessing misc -Butanol misc Protein engineering
topic_browse	misc Cellulosic biofuel misc Consolidated bioprocessing misc -Butanol misc Protein engineering
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title	Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose
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title_full	Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose
author_sort	Tian, Liang
journal	Biotechnology for biofuels
journalStr	Biotechnology for biofuels
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author_browse	Tian, Liang Conway, Peter M. Cervenka, Nicholas D. Cui, Jingxuan Maloney, Marybeth Olson, Daniel G. Lynd, Lee R.
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author-letter	Tian, Liang
doi_str_mv	10.1186/s13068-019-1524-6
normlink	(ORCID)0000-0002-6419-3695
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title_sort	metabolic engineering of clostridium thermocellum for n-butanol production from cellulose
title_auth	Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose
abstract	Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. © The Author(s) 2019
abstractGer	Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. © The Author(s) 2019
abstract_unstemmed	Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. © The Author(s) 2019
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title_short	Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose
url	https://dx.doi.org/10.1186/s13068-019-1524-6
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author2	Conway, Peter M. Cervenka, Nicholas D. Cui, Jingxuan Maloney, Marybeth Olson, Daniel G. Lynd, Lee R.
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