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 sustainabilit...

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