S85: Metabolic Engineering Of c. Thermocellum For Biofuel Production From Cellulose

Clostridium thermocellum is a leading candidate organism for implementing a consolidated bioprocessing (CBP) strategy due to its native ability to rapidly consume cellulose and its existing ethanol production pathway. Since substrate costs are a significant fraction of final costs, high product yield is needed for commercial viability. C. thermocellum converts cellulose and cellobiose to lactate, formate, acetate, hydrogen, ethanol, amino acids, and other products. However, the mechanism for flux distribution at the various metabolic branch points is not well understood. Furthermore, while pyruvate kinase typically converts phosphoenolpyruvate (PEP) to pyruvate during glycolysis, it is absent from the C. thermocellum genome sequence. The lack of pyruvate kinase leads to the hypothesis that C. thermocellum utilizes an unusual glycolytic pathway in which PEP carboxykinase converts PEP to oxaloacetate, malate dehydrogenase converts oxaloacetate to malate, and malic enzyme converts malate to pyruvate. To better understand central metabolism and to increase ethanol yield, we have constructed C. thermocellum deletion and overexpression mutants to constrain flux of carbon and electrons through glycolysis. Multiple approaches are being undertaken, including alteration of alcohol dehydrogenase cofactor specificity, deletion of genes involved in production of H2, acetate, lactate, and formate, and strain evolution. The resulting strains produce substantially more ethanol than the wild type and are being further modified by both rational and random approaches. Phenotypic and genotypic 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 lignocellulosic biomass.