Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature
Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hy- perthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.
- Research Organization:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC05-00OR22725; AC36-08GO28308
- OSTI ID:
- 1433727
- Alternate ID(s):
- OSTI ID: 1240243; OSTI ID: 1327708
- Report Number(s):
- NREL/JA-2700-65970; S2214030116300049; PII: S2214030116300049
- Journal Information:
- Metabolic Engineering Communications, Journal Name: Metabolic Engineering Communications Vol. 3 Journal Issue: C; ISSN 2214-0301
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- Netherlands
- Language:
- English
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