Thermophilic deconstruction of plant biomass by Caldicellulosiruptor bescii

Thermophilic organisms and their enzymes offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. The use of complex feedstocks 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, such as ethanol. 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 populus and switchgrass and do so in the absence of a cellulosome. We have used genetic methods developed in our lab to generate deletions of genes predicted to be involved in biomass utilization including celA, a family-48/family-9 glycosyl hydrolase and a cluster of genes involved in pectin utilization. These mutants are dramatically reduced in their ability to deconstruct plant cell walls and we report their use to identify key plant cell wall components that contribute to plant biomass recalcitrance. We have also introduced endoglucanases and xylanases from the Gram-positive thermophilic actinomycete, Acidothermus cellulolyticus and show that their expression greatly improves the C. bescii exoproteome. The mechanism of this synergy reveals important aspects of enzyme function.