SBIR Phase II: Industrial Strength Biology: Creating Robust Protein Technologies Using Spore-Display Directed Evolution

The broader/commercial impact of this Small Business Innovation Research (SBIR) Phase II project is to provide a novel synthetic biology platform that generates customizable enzyme solutions for industrial biocatalyst applications. The use of enzymes as industrial biocatalysts continues to expand, offering economically viable and non-toxic solutions to a wide range of industrial processes. Viewed as an alternative to conventional chemical catalysts, enzyme biocatalysts offer greater safety in their processes owing to their biodegradable nature, high selectivity, ability to operate under mild reaction conditions, and their ability to generate a low amount of byproduct during a reaction; they also negate the need for potentially toxic, energy intensive, and costly reagents typically needed for conventional chemical catalysis. With the proposed technology’s enhanced capabilities, there is potential to increase this impact by providing novel enzyme solutions that confer greater robustness and efficiency at lower costs. The proposed project aims to develop a synthetic biology platform that leverages a process called spore-display immobilization, in which bacteria make and assemble enzymes on the surface of spores, a self-assembling and genetically encoded microparticle. While enzyme catalysis is used in a wide range of industries, the ability to create enzymes with thermal and chemical stability that are also recyclable remains a challenge. By applying directed evolution and high-throughput screening technologies to spore-displayed enzymes, the platform enables rapid prototyping of spore-enzyme variants to improve important variables like enzyme activity, stability, and loading density. Phase I used lipases to create a spore-displayed lipase variant with an optimal operating temperature 20 °C higher than the soluble parent enzyme. This established that the directed evolution of spore-displayed proteins is possible, laying the groundwork for the development of many spore-displayed protein products. To further develop this technology, Phase II has the following objectives: (1) Increasing the half-life of spore-displayed enzymes through genetic code expansion; (2) Enabling spore-display on the spores of bacteria other than Bacillus subtilis that have industrially-relevant phenotypes; and (3) Developing next-generation technologies that facilitate low-cost separation of spores from reaction products. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.