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

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 . To develop more thermostable genetic tools for , we constructed vectors based on the hyperthermophilic replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from were functional in . 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.