S116: Genetic tool development and metabolic engineering of C. thermocellum for biofuel production

Clostridium thermocellum is a thermophilic anaerobic bacterium that specializes in the rapid solubilization and fermentation of crystalline cellulose to products that include ethanol. As such, it is considered a prime candidate for consolidated bioprocessing, the process by which a single organism is responsible both for the hydrolysis and fermentation of plant biomass for biofuel production. However, the nascent genetic tools limit the speed and reliability of genetic modification. To remedy this situation, we have examined the effect of DNA methylation on transformation, increasing transformation efficiency up to three orders of magnitude. This and other advances in genetic tool development in this organism have greatly accelerated metabolic engineering efforts to reroute central metabolism toward biofuel production. Furthermore, recent work on closing the carbon balance in C. thermocellum has suggested a possible redox imbalance limits ethanol production. To address this possibility, the new tools have been utilized to construct C. thermocellum deletion mutants to constrain carbon and electron flux, resulting in strains that produce substantially more ethanol than the wild type. Phenotypic characterization of these strains gives insight into central metabolism of C. thermocellum and suggests future paths for the engineering of more efficient biofuel production from plant biomass.