2-41: Methylation in vitro and in vivo enables DNA transformation of Caldicellulosiruptor species: use for metabolic engineering and direct conversion of biomass to biofuels and chemicals

Thermophilic organisms offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. The use of these complex substrates often requires pretreatment, involving exposure to acid or base at high temperature and the addition of hydrolytic enzymes that partially digest the plant cell walls. Enzymatic pretreatment is expensive and often prohibitive for the production of low value commodity products. 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. Members of this genus vary in their ability to use unpretreated substrates including switchgrass. The ability to genetically manipulate these organisms is a prerequisite for engineering them for use in conversion of complex substrates to products of interest. Here we report methods for efficient DNA transformation of members of this genus. We show that restriction of DNA is a major barrier to transformation and present methods to overcome it. We have used this genetic system to construct deletions of genes predicted to be involved in biomass utilization as well as construct pathways for fuel production.