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 use of 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.
Increasing our ability to efficiently use cellulosic biomass as a source of simple sugars will play a key role in enhancing energy sustainability as it will reduce our dependence on oil by facilitating the bio-based production of transportation fuels, chemicals, and other products that are currently derived from petroleum. A major obstacle in the production of renewable fuels and chemicals is the inefficiency of enzymatic hydrolysis—the process used to convert cellulosic biomass to simple sugars. The inefficiency stems in part from the fact that lignocellulose is extremely recalcitrant to degradation and thus requires large quantities of multiple synergistic enzymes to break it down, making the process rather costly. Protabit’s strategy to reduce the cost is to engineer more efficient thermostable enzymes. Creating heat-resistant cellulolytic enzymes will increase enzyme lifetimes and allow reactions to proceed at higher temperatures. These factors will decrease the amount of enzyme and the processing time required per unit of feedstock, leading to significant cost savings. Our overall goal was therefore to improve the thermostabilities of a set of key cellulolytic enzymes, thereby enhancing their ability to release sugars from cellulosic biomass at elevated temperature. However, helper enzymes called polysaccharide monooxygenases (PMOs) were recently discovered. These enzymes act by disrupting the crystalline nature of cellulose, making the individual fibers more accessible to the cellulolytic enzymes that digest them. When used in conjunction with cellulolytic enzymes, PMOs can significantly increase the overall degradation of biomass. In light of these findings, we extended our goals to include obtaining a thermostable PMO to add to our mix of thermostabilized cellulolytic enzymes. The focus of this Phase I project was therefore to identify and/or engineer a highly thermostable PMO. In collaboration with the Mayo lab at Caltech, we characterized seven different natural PMOs for expression, catalytic activity, thermostability, and ability to enhance cellulose degradation of a 3-enzyme cocktail, and identified the best ones for engineering. We then applied computational protein design (CPD) to enhance the thermostability of the target PMOs, resulting in 14 combinatorial libraries of engineered PMO sequences. The initial PMO libraries have been expressed and experimental high-throughput screening is in progress. In addition, in the course of this project, substantial improvements were made in automating our assays and design-engineering workflow, resulting in up to a 100-fold increase in throughput. We also developed a "heat challenge" assay that is particularly important because it allows us to bypass the bottleneck that occurs when some of the key enzymes in the cocktail are not fully thermostabilized. In previous work, funded in part by NSF, we engineered variants of a key cellulase (the endoglucanase Cel5A) that are stabilized by up to 16°C. Including one of these thermostabilized variants in a 3-enzyme cocktail significantly improved sugar yields from pretreated corn stover, allowing a 3-fold reduction in enzyme loading at elevated temperatures. Importantly, these results demonstrate the validity of our strategy of improving the efficiency of enzymatic hydrolysis via enzyme stabilization. We have also made progress toward thermostabilizing two other key cellulases. These accomplishments and the improvements in our protein engineering platform have put us in a great position to proceed with our plans for Phase II: to continue engineering PMOs and other key enzymes to have improved thermostability and cellulose-degrading activity, and to validate them in lab- and pilot-scale ethanol fermentation trials. In addition to accelerating U.S. progress toward economic, energy, and environmental sustainability via the more efficient degradation of biomass to sugars, this project has additional merit in its use of CPD-based protein engineering. These "in silico" methods significantly reduce the cost of research by shifting a substantial amount of experimental screening effort to the software platform. The designed libraries output by CPD are typically enriched in functional variant sequences, accelerating the speed of obtaining functional proteins with the desired properties. This research has also contributed to improving Protabit’s CPD-based protein engineering platform, thereby making it more useful for protein stabilization and protein design in general. These improved tools will enhance the ability of investigators to design proteins for biofuels production and to solve protein-engineering problems in general in the industrial, medical, and agricultural biotechnology fields.
