Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)
Abstract
Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.
- Authors:
- Publication Date:
- Research Org.:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1250131
- Alternate Identifier(s):
- OSTI ID: 1286889
- Grant/Contract Number:
- AC05–00OR22725; AC02–05CH11231; AC05-00OR22725
- Resource Type:
- Published Article
- Journal Name:
- Metabolic Engineering Communications
- Additional Journal Information:
- Journal Name: Metabolic Engineering Communications Journal Volume: 3 Journal Issue: C; Journal ID: ISSN 2214-0301
- Publisher:
- Elsevier
- Country of Publication:
- Netherlands
- Language:
- English
- Subject:
- 60 APPLIED LIFE SCIENCES
Citation Formats
Hon, Shuen, Lanahan, Anthony A., Tian, Liang, Giannone, Richard J., Hettich, Robert L., Olson, Daniel G., and Lynd, Lee R. Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs). Netherlands: N. p., 2016.
Web. doi:10.1016/j.meteno.2016.04.001.
Hon, Shuen, Lanahan, Anthony A., Tian, Liang, Giannone, Richard J., Hettich, Robert L., Olson, Daniel G., & Lynd, Lee R. Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs). Netherlands. https://doi.org/10.1016/j.meteno.2016.04.001
Hon, Shuen, Lanahan, Anthony A., Tian, Liang, Giannone, Richard J., Hettich, Robert L., Olson, Daniel G., and Lynd, Lee R. Thu .
"Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)". Netherlands. https://doi.org/10.1016/j.meteno.2016.04.001.
@article{osti_1250131,
title = {Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)},
author = {Hon, Shuen and Lanahan, Anthony A. and Tian, Liang and Giannone, Richard J. and Hettich, Robert L. and Olson, Daniel G. and Lynd, Lee R.},
abstractNote = {Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.},
doi = {10.1016/j.meteno.2016.04.001},
journal = {Metabolic Engineering Communications},
number = C,
volume = 3,
place = {Netherlands},
year = {Thu Dec 01 00:00:00 EST 2016},
month = {Thu Dec 01 00:00:00 EST 2016}
}
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https://doi.org/10.1016/j.meteno.2016.04.001
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Works referencing / citing this record:
Engineering Clostridium for improved solvent production: recent progress and perspective
journal, May 2019
- Cheng, Chi; Bao, Teng; Yang, Shang-Tian
- Applied Microbiology and Biotechnology, Vol. 103, Issue 14
Engineering Clostridium for improved solvent production: recent progress and perspective
journal, May 2019
- Cheng, Chi; Bao, Teng; Yang, Shang-Tian
- Applied Microbiology and Biotechnology, Vol. 103, Issue 14
A mutation in the AdhE alcohol dehydrogenase of Clostridium thermocellum increases tolerance to several primary alcohols, including isobutanol, n-butanol and ethanol
journal, February 2019
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Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase
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Expression of adhA from different organisms in Clostridium thermocellum
journal, November 2017
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- Biotechnology for Biofuels, Vol. 10, Issue 1
Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production
journal, September 2018
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- Biotechnology for Biofuels, Vol. 11, Issue 1
Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production
journal, March 2020
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- Biotechnology for Biofuels, Vol. 13, Issue 1