P96: Development of Genetic Systems in Clostridium thermocellum

Clostridium thermocellum is a thermophilic anaerobic bacterium that can break down cellulose and generate hydrogen (H2) and ethanol. This makes it an ideal organism in the conversion of lignocellulosic biomass to clean and renewable biofuels. Analysis of its genome sequencing revealed that C. thermocellum harbors four putative hydrogenase enzymes (three FeFe-H2ases and one NiFe-H2ase) involved in H2 catalysis. Under fermentative conditions C. thermocellum also produces formate, ethanol, lactate, and acetate in addition to H2. At present this microbe is very recalcitrant to genetic manipulations. We thus attempted to develop a genetic system using several methods such as conjugation, electroporation and autoplast transformation with varying degree of success. We thus carried out metabolic pathway inhibitor experiments to determine effects of blocking competing pathway on H2 production. Our studies on blocking the formate production pathway by chemical inhibitor sodium hypophosphite resulted in an overall increase in H2 production, which parallels an alteration in the profiles of other metabolites including lactate, acetate, ethanol, and formate. An increase in hydrogenase activity was observed when formate biosynthesis was blocked, suggesting regulations in hydrogenase gene expression. Our findings suggest that knock out of pyruvate formate-lyase (PFL) by genetic recombination would increase the H2 production similar to the level of the chemical inhibitor. Collectively, our results indicate that H2 production can be significantly enhanced via blocking competing pathways during cellulose metabolism.