S117: Methylation by cognate methyltransfersases is required for DNA transformation of Caldicellulosiruptor species: construction of mutants for the conversion of biomass to biofuels

Thermophilic organisms offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65 °C to 78 °C and are the most thermophilic cellulolytic organisms known. In fact, C. bescii is capable of using unpretreated biomass, including switchgrass for growth. The ability to genetically manipulate these organisms is a prerequisite for engineering them for use in conversion of these complex substrates to products of interest. Here we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation and that methylation of heterologous DNA with a unique α-class N4-Cytosine methyltransfersase is required for DNA transformation. We have extended this genetic methodology to other members of this genus and have begun the construction of pathways for the metabolic engineering of biomass to biofuels. We have also constructed mutants that allow the elucidation of the mechanism used by members of this group degrade unprocessed biomass.