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The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus  Caldicellulosiruptor  are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for  Caldicellulosiruptor species  based on pBAS2, the smaller of two native  C. bescii  plasmids. The entire plasmid was cloned into an  E. coli  cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in  E. coli . The wild-type  C. bescii pyrF  locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105), and the resulting vector was transformed into a new spontaneous deletion mutant in the  pyrFA  locus of  C. bescii  that allowed complementation with the  pyrF  gene alone. Plasmid DNA was methylated  in vitro  with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in  C. bescii . A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of  C. hydrothermalis,  an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the  C. bescii celA  gene were maintained with selection and were structurally stable through transformation and replication in  C. bescii  and  E. coli .

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Construction of a Stable Replicating Shuttle Vector for Caldicellulosiruptor Species: Use for Extending Genetic Methodologies to Other Members of This Genus