This Small Business Innovation Research Phase I project aims to apply Protabit?s state-of-the-art computational protein design (CPD) platform to engineer a more thermostable and cost-effective set of lignocellulosic enzymes for converting biomass to simple sugars. CPD is an innovative technology that yields enzymes with novel or enhanced properties that cannot be found using traditional protein engineering methods, and Protabit is actively developing the most advanced, commercial-grade CPD platform available. In Phase I, Protabit and the Mayo Lab at the California Institute of Technology will stabilize two important commercial cellulases, H. jecorina Cel5A and Cel7A. The objectives of Phase I are: (a) to apply CPD to design libraries of thermostable Cel5A and Cel7A variants, and to use high-throughput screening to identify variants with optimal cellulolytic activity at elevated temperatures; (b) to measure the degree to which the most active thermostable variants reduce enzyme loading and hydrolysis time on pretreated corn stover, and to estimate the corresponding cost savings; and (c) to characterize the most active thermostable variants for other commercially important properties such as pH profile and expression yield. In Phase II, Protabit and Caltech will similarly engineer other key lignocellulosic enzymes and optimize this core set for activity on corn stover, switchgrass, and other biomass feedstocks.

The broader impacts/commercial potential of this research are: (a) to reduce the costs of converting biomass into glucose and other simple sugars, which are a principal raw material input in the rapidly growing renewable fuels and chemicals industries, and (b) to demonstrate Protabit's versatile protein engineering platform technology on a specific problem of commercial significance in industrial biotechnology. By facilitating the bio-based production of advanced drop-in biofuels, ethanol, and precursors for synthetic rubber, plastics, and other petroleum-derived materials, this research can help reduce U.S. dependence on foreign oil and spur domestic manufacturing, investment, and job creation. In addition, sourcing sugars from cellulose, the most abundant polymer on the planet, can curb the food-versus-fuel debate by reducing demand for edible corn as a biofuels feedstock; it also encourages the farming of dedicated feedstock crops capable of growing on marginal lands unsuitable for food production. Furthermore, CPD-based protein stabilization methods enable new products and technologies in myriad areas, including industrial enzymes, protein materials, novel antibody-like scaffolds, and therapeutics with improved shelf-life or biological half-life. With this project, Protabit and Caltech will help the U.S. take a major step toward economic, energy, and environmental security.

Project Report

Efficiently sourcing fermentable sugars from lignocellulosic biomass will play a critical role in enhancing energy sustainability by reducing our dependence on petroleum for the production of transportation fuels and other products. It will also curb the food-versus-fuel debate by reducing demand for edible biomass (corn and sugarcane) as a biofuels feedstock. A major hurdle in the production of renewable fuels and chemicals is the inefficiency of enzymatic hydrolysis—the process used to convert cellulosic biomass to simple sugars. This is due in part to the inaccessibility and recalcitrance of lignocellulose and to the high cost of the multiple enzymes required to degrade it. Protabit’s approach is to reduce the cost of enzymatic hydrolysis by engineering more efficient thermostable cellulolytic enzymes. Creating heat-resistant 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. The overall goal is 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. Phase I of this NSF-funded project was recently completed and has yielded excellent results. Using computational protein design (CPD) and high-throughput screening, Protabit and the Mayo lab at the California Institute of Technology increased the thermostability of a key hydrolytic enzyme (the endoglucanase Cel5A) by a dramatic 9°C. Including this thermostabilized variant 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 also demonstrated the validity of our strategy of improving the efficiency of hydrolysis via enzyme stabilization. We hope to continue these studies in Phase II and engineer other key cellulolytic enzymes to have improved thermostability and biomass-degrading activity. 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 methods. These "in silico" methods significantly reduce the cost of biological research by shifting a substantial amount of laboratory screening effort to the software platform. The variant libraries generated by CPD are typically enriched in functional variant sequences, accelerating the process of obtaining functional proteins with the desired properties. This Phase I project has also contributed to improving the algorithms in Protabit’s CPD software suite, thereby making it more useful for protein stabilization and protein engineering in general. The improved software will enable investigators to more readily engineer useful proteins for industrial, medical, and agricultural biotechnology applications.

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Protabit LLC
United States
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