Extending Genetic Methods to Members of the Anaerobic, Thermophilic Genus Caldicellulosiruptor for the Conversion of Plant Biomass to Biofuels

Decomposition of complex cell wall polysaccharides is the major bottleneck in the conversion of plant biomass to biofuels and chemicals. Conventional biomass pretreatment includes exposure to high temperatures, acid or base as well as enzymatic digestion. Members of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms known. They grow optimally at 65-78°C and degrade lignocellulosic biomass without conventional pretreatment. C. hydrothermalis is of special interest because it lacks the native high copy plasmid found in the related species C. bescii, making the shuttle vector based on this plasmid a high copy number expression vector. It is also among the least cellulolytic in comparison to other species, making it an ideal naïve system to study key cellulolytic enzymes. In order to make C. hydrothermalis genetically tractable, we screened for a spontaneous deletion of the pyrimidine biosynthesis gene pyrF.  Deletion of this gene resulted in uracil auxotrophy and resistance to 5-fluoroorotic acid (5-FOA), allowing prototrophic selection of transformants and counter-selection of the wild-type allele. The C. hydrothermalis ΔpyrF mutant was transformed with a shuttle vector using the wild-type pyrF allele from Clostridium thermocellum as a selectable marker.  This strain maintains the shuttle vector at ~50 copies per cell. To increase transformation efficiency we deleted the gene encoding ChyI, the homolog of a restriction enzyme shown to be an absolute barrier to transformation in C. bescii. The C. hydrothermalis ΔpyrF ΔchyI strain will enable the assessment of biomass utilization enzymes from other Caldicellulosiruptor species.