Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)

Shuen Hon; Anthony A. Lanahan; Liang Tian; Richard J. Giannone; Robert L. Hettich; Daniel G. Olson; Lee R. Lynd

doi:10.1016/j.meteno.2016.04.001

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)

Shuen Hon, Anthony A. Lanahan, Liang Tian, Richard J. Giannone, Robert L. Hettich, Daniel G. Olson, Lee R. Lynd

Bioanalytical Mass Spectrometry

Research output: Contribution to journal › Article › peer-review

19 Scopus citations

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.

Original language	English
Pages (from-to)	120-129
Number of pages	10
Journal	Metabolic Engineering Communications
Volume	3
DOIs	https://doi.org/10.1016/j.meteno.2016.04.001
State	Published - Dec 2016

Funding

The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research (Grant no. DE-AC05–00OR22725) in the DOE Office of Science. The manuscript has been authored by Dartmouth College under sub-contract no. 4000115284 and contract no. DE-AC05–00OR22725 with the U.S. Department of Energy. Resequencing was performed by the Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, and is supported by the Office of Science of the U.S. Department of Energy under contract number DE-AC02–05CH11231. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research (Grant no. DE-AC05–00OR22725 ) in the DOE Office of Science. The manuscript has been authored by Dartmouth College under sub-contract no. 4000115284 and contract no. DE-AC05–00OR22725 with the U.S. Department of Energy. Resequencing was performed by the Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, and is supported by the Office of Science of the U.S. Department of Energy under contract number DE-AC02–05CH11231 . Appendix A

Funders	Funder number
BioEnergy Science Center
DOE Office of Science user facility
Department of Energy Joint Genome Institute
Office of Biological and Environmental Research
U.S. Department of Energy Bioenergy Research Center
U.S. Department of Energy	DE-AC02–05CH11231
Office of Science
Biological and Environmental Research
Dartmouth College	4000115284, DE-AC05–00OR22725

Keywords

Clostridium Thermocellum
Gene expression
Plasmid
Structural stability
adhE

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10.1016/j.meteno.2016.04.001

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title = "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.",

keywords = "Clostridium Thermocellum, Gene expression, Plasmid, Structural stability, adhE",

author = "Shuen Hon and Lanahan, {Anthony A.} and Liang Tian and Giannone, {Richard J.} and Hettich, {Robert L.} and Olson, {Daniel G.} and Lynd, {Lee R.}",

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year = "2016",

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AU - Tian, Liang

AU - Giannone, Richard J.

AU - Hettich, Robert L.

AU - Olson, Daniel G.

AU - Lynd, Lee R.

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N2 - 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.

AB - 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.

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Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)

Abstract

Funding

Keywords

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