Towards cellulosic ethanol: bioengineering of cellulases

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The rate limiting step in the conversion of cellulosic biomass to ethanol is the efficient release of glucose from feedstocks. The crystallinity of the substrate, the high inherent stability of the glycosidic bond, and the presence of inhibitory materials, such as lignin fragments, furfurals, and other polyphenolics, in the pre-treated biomass all pose challenges and serve to drive up costs for its bioconversion. Indeed, a joint study by the U.S. Department of Energy and the U.S. Department of Agriculture estimated that the cost of production of ethanol from lignocellulosics is approximately double that for the same production from starch sources, such as corn. The experimental plan presented in this proposal represents a continuation of studies on cellulolytic and related enzymes conducted in our laboratory over the past 24 years under the auspices of NSERC. Our objective is to reduce the cost of pre-treatment of lignocellulosic biomass by engineering cellulolytic enzymes with enhanced properties. To this end, we propose to: 1) investigate the general applicability of replacing the general base catalytic residues of an inverting enzyme with a sulfinate functional group to modulate the catalytic potential and pH profile of cellulolytic enzymes; 2) continue both the rationale and random mutagenesis of C. fimi CenA and CbhA genes to generate cellulolytic enzymes with more acidic pH-activity profiles; and 3) screen mutants generated by random mutagenesis for altered pH activity profiles. We also propose to test the hypothesis that the carbohydrate-binding modules of some cellulolytic enzymes possess the ability to disrupt the crystallinity of cellulose non-hydrolytically. These binding modules will be produced independent of their parent enzyme and their ability to disrupt the structure of crystalline cellulose will be observed in real time by atomic force microscopy. Attempts to enhance this non-hydrolytic activity will be made by site-directed mutagenesis of the encoding genes to produce modules with specific amino acid replacements. Finally, I propose to continue our basic studies on the structure and function relationship of glycoside hydrolases by attempting to convert an inverting enzyme into on that retains the anomeric configuration of the substrate in the hydrolytic product.