Pathway engineering to improve ethanol production by thermophilic bacteria

Abstract

The Project Summary should include a statement of objectives, methods to be employed, and the significance of the proposed activity to the advancement of knowledge or education. Avoid use of first person to complete this summary. DO NOT EXCEED ONE PAGE. (Some Programs may impose PATHWAY ENGINEERING TO BYA PROJECT SUMMARYContinuation of a research project jointly funded by the N,SF and DOE is proposed. The primary project goal is to develop and characterize strains of C. thermocellum and C. themosaccharolyticum having ethanol selectivity similar to more conventional ethanol-producing organisms. An additional goal is to document the maximum concentration of ethanol that can be produced by thermophiles. ' These goals build on results from the previous project, including development of most of the genetic tools required for pathway engineering in the target organisms. As well, we demonstrated that the tolerance of C. thermosaccharolyticum to added ethanol is sufficiently high to allow practical utilization should similar tolerance to produced ethanol be demonstrated, and that inhibition by neutralizing agents may explain the limited concentrations of ethanol produced in studies to date.Task 1 involves optimization of electrotransformation, using either modified conditions or alternative plasmids to improve upon the ,low but reproducible transformation frequencies we have obtained I thus far. Task 2 involves cloning target thermophilic genes (those encoding for acetate kinase, phospohotransacetylase, lactate dehydrogenase, hydrogenase, and pyruvate dehydrogenase from C. thermocellum and C. thermosaccharolyticum) in E. coli. Task 3 consists of preparing plasmid constructs containing deleted copies of target genes, introducing these constructs into thermophilic hosts via electroporation, and selection of strains in which non-functional deleted genes have replaced functional target genes via homologous recombination. In Task 4, fermentation studies will be carried out directed toward realizing the ethanol production potential of thermophiles and characterization of modified strains produced in Task 3. Project results are expected to be significanr. in the context of: 1) advancing technology for production of ethanol, a sustainable fuel and chemical feedstock of considerable promise; 2) demonstrating the efficacy of consolidated bioprocessing, a process concept generically applicable and potentially highly advantageous for low cost productim of commodity products from biomass; and 3) extending pathway (or metabolic) engineering by applying it to anaerobic thermophilic bacteria, a potentially important group of microorganisms for which molecular-level tools and information are not well-developed. DISCLAIMERThis report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or u...