STTR Phase I: Engineering Polysaccharide Monooxygenases for Enhanced Sugar Recovery From Biomass

This Small Business Technology Transfer (STTR) Phase I project aims to apply computational protein design (CPD) to engineer polysaccharide monooxygenases (PMOs) for improved thermostability, so that a cocktail of PMOs and key cellulases will more efficiently convert lignocellulolosic biomass to fermentable sugars. The objectives of Phase I are to: (a) evaluate several natural PMOs for expression, thermostability, PMO activity, and ability to enhance the cellulolytic activity of a cellulase cocktail, and then select one or more as targets for engineering; (b) use CPD to design the PMOs for improved thermostability; (c) experimentally screen libraries of designed PMO variants; and (d) improve the properties of best hits with additional rounds of engineering. The work proposed here will help elucidate the role PMOs play in cellulolytic degradation in the context of other cellulases. This project may further our knowledge of the substrate specificities of these enzymes. Any novel crystal structures that may be solved could also enhance our understanding of the structural differences and substrate specificities within this family of enzymes and may serve to elucidate structure-function relationships. The anticipated technical result is one or more PMO variants that have increased thermostability of 5-10°C relative to the wild-type PMO. The broader impact/commercial potential of this project, if successful, will be to: (a) reduce the costs of converting biomass into simple sugars, which are a principal raw material for the production of renewable fuels and chemicals, (b) encourage the broader useof enzymatic hydrolysis, a sustainable and environmentally-friendly process, and (c) demonstrate the utility of our protein engineering platform. By facilitating the bio-based production of ethanol, advanced drop-in biofuels, and substitutes for other petroleum-derived materials, this research can help reduce U.S. dependence on foreign oil, reduce our carbon footprint, and spur domestic manufacturing, investment, and job creation. In addition, sourcing sugars from cellulose can curb the food-versus-fuel debate by encouraging the farming of dedicated feedstock crops capable of growing on marginal lands unsuitable for food production. CPD-based protein engineering methods can significantly reduce the costs of biological research by shifting a significant amount of experimental screening effort to the software platform. The designed libraries output by CPD are typically enriched in functional variants, accelerating the delivery of functional end-products. Furthermore, CPD-based protein stabilization methods enable new products and technologies in myriad areas, including industrial enzymes and therapeutics. This project will accelerate U.S. progress toward economic, energy, and environmental sustainability.