Abstract
Background: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. Results: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+dcm+E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. Conclusions: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.
| Original language | English |
|---|---|
| Article number | 30 |
| Journal | Biotechnology for Biofuels |
| Volume | 5 |
| DOIs | |
| State | Published - 2012 |
Funding
This work was supported in part by the BioEnergy Science Center at Oak Ridge National laboratory. The BioEnergy Science Center is a U.S. DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. Additional support was provided by the DOE Energy Biosciences Program Grant DE-FG02-02ER1535, and Mascoma Corporation. We are grateful to Chris Herring and Anne Warner of Mascoma Corporation for sharing information on plasmid pB6A prior to publication, and to Marybeth Maloney and Jessica Whitaker of Dartmouth College for technical assistance.
| Funders | Funder number |
|---|---|
| BioEnergy Science Center | |
| DOE Energy Biosciences Program | DE-FG02-02ER1535 |
| Mascoma Corporation | |
| Office of Biological | |
| U.S. Department of Energy | DE-AC05-00OR22725 |
| Oak Ridge National Laboratory |
Keywords
- Clostridium thermocellum
- Consolidated bioprocessing
- DNA methylation
- Transformation efficiency
