Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum
- ORNL
- Thayer School of Engineering at Dartmouth
- Mascoma Corporation
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 pAM205 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. CONCLUSION: 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.
- Research Organization:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- DE-AC05-00OR22725
- OSTI ID:
- 1049799
- Journal Information:
- Biotechnology for Biofuels, Vol. 5, Issue 30
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
BEARINGS
BIOFUELS
CLOSTRIDIUM THERMOCELLUM
DNA
EFFICIENCY
ESCHERICHIA COLI
GENES
GENETICS
METHYLATION
MICROORGANISMS
MUTATIONS
PLASMIDS
PRODUCTION
REMOVAL
TRANSFORMATIONS
Clostridium thermocellum
DNA methylation
Transformation efficiency
Consolidated bioprocessing