Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

• Intergeneric replication of a hyperthermophilic plasmid replicon in C. thermocellum.
• Transformation efficiencies >3000 colony forming units per µg of plasmid DNA.
• Plasmid maintained at ~20 copies per chromosome in C. thermocellum.
• Caldicellulosiruptor proteins and promoters function in C. thermocellum.
• The pBAS2 RepL replication initiation protein is unique among thermophilic proteins.

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 hyperthermophilic 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. These results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.