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INVESTIGATOR: O`Malley, M. A.

PERFORMING INSTITUTION:
Massachusetts Institute of Technology
Cambridge, MASSACHUSETTS 02139

GENETIC IDENTIFICATION AND CHARACTERIZATION OF CELLULASES AND CELLULOLYTIC COMPLEXES FROM FUNGI

NON-TECHNICAL SUMMARY: The energy contained within plant biomass is the most promising renewable resource available for biofuel development, and is an attractive alternative to petroleum-based fuels. Most of the energy in plant material is contained within cellulose, a complex polymer of sugars in plant cell walls, which can be broken down into simple sugars for fermentation into energy-rich organic fuels (e.g. ethanol). However, the complicated structure of cellulose renders this approach quite challenging due to difficulties associated with enzymatic conversion of cellulose to simple sugars. Fortunately, nature has evolved several enzymes over millions of years that work together to break down plant material in this fashion. These enzymes can be found within bacteria and fungi that thrive in cellulose-rich environments (e.g., the digestive tract of grazing animals, compost piles, and soil). In order to accelerate the development of plant-based biofuels, this project aims to discover new cellulolytic enzymes from fungi that reside within the digestive tract of large herbivores, and to exploit them for bioprocessing. Novel enzymes will be annotated based on analyzing real-time protein production within the fungi when supported on plant material in isolated culture. Attractive cellulolytic enzymes will be produced in Saccharomyces cerevisiae (baker's yeast) to screen their reactivity and optimize their performance against plant biomass. This project will serve to significantly expand the number and diversity of known enzymes to facilitate industrial-scale cellulose breakdown, and will improve the economic feasibility of plant-based biofuels.

OBJECTIVES: This project aims to accelerate the development of cellulosic biofuels by applying powerful genomic and biochemical tools towards the discovery of novel cellulose-degrading enzymes that originate from fibrolytic anaerobic fungi. These fungi reside within the rumen and hindgut of large herbivorous animals that naturally thrive on a lignocellulose-rich diet, yet remarkably few enzymes have been identified and characterized from these microbes. Specific objectives for this project are to: (1) develop a new molecular genetic platform to identify novel fungal cellulases by illuminating differences in relative transcription levels when grown on varied carbon sources; (2) screen and evaluate activity of anaerobic fungal cellulases identified via catabolic regulation studies through heterologous expression in the yeast S. cerevisiae; and (3) optimize catalytic performance of fungal cellulases expressed in yeast to achieve maximal lignocellulase breakdown for fermentation into ethanol. Expected experimental outcomes from this project include the creation of a novel bioinformatics-based platform for cellulase discovery and the isolation of full DNA coding sequences for non-catalytic adapter complexes and cellulolytic enzymes from anaerobic fungi. Using this information, combinatorial yeast strains harboring recombinant fungal cellulases for synergistic action against lignocellulose will also be constructed and evaluated to facilitate Consolidated BioProcessing (CBP).