Grant: Strategic Projects Supplemental Competition

The long-term objective of this research is to develop novel strains of cellulolytic bacteria with enhanced ability to synthesize biofuels (ethanol or hydrogen) directly form cellulosic feedstocks. Our model organism, Clostridium thermocellum, is a gram-positive, thermophilic, acetogenic, anaerobe that can utilize cellulose as a sole carbon source and synthesizes ethanol, acetate, and other organic acids via a mixed fermentation metabolism, with the concomitant release of hydrogen (H2) and carbon dioxide (CO2). The ability of C. thermocellum to utilize cellulosic substrates for ethanol or H2 synthesis offers excellent potential for the practical use of this bacterium for biofuels production via consolidated bioprocessing, whereby simultaneous substrate hydrolysis and fermentation of the substrate are carried out by the microorganism without (or with minimal) feedstock pre-treatment. Consolidated bioprocessing involves simultaneous saccharification and fermentation, and offers the potential for lower biofuel production costs due to simpler feedstock processing, lower energy inputs (and therefore better energy balance), and higher conversion efficiencies than current bioethanol production processes that utilize a process of separate substrate hydrolysis, followed by fermentation. Consolidated bioprocessing is an economically attractive near-term goal for "third generation" biofuel production. We will create strains of C. thermocellum with enhanced capability for ethanol or H2 production by knocking-down transcription of genes that encode key genes that mediate pyruvate catabolism, or by completely knocking-out expression of these target genes. The specific objectives of this proposal are to: 1) develop an efficient transformation protocol for C. thermocellum; 2) construct, evaluate, and utilize knock-down and knock-out vectors to selectively alter metabolic fluxes in C. thermocellum cultured on cellulosic feedstocks; 3) evaluate changes in end-product synthesis patterns, gene transcription, and selected gene product expression profiles in recombinant C. thermocellum.