Directed evolution of immobilized enzymes on bacterial spores

Enzymes catalyze chemical reactions. They can improve chemical processes, but there are major challenges to realizing their full potential. Most chemical processes involve harsh conditions. These can be high temperatures, extreme pHs, and the presence of organic solvents. This project will identify enzymes that work under harsh conditions by exploiting two characteristics of spores. Spores are highly resistant to harsh conditions, and they can be engineered to display proteins on their surfaces. The experimental strategy will be to generate a large number of mutated enzymes that are displayed on spore surfaces. The spores will then be subjected to a variety of harsh conditions mirroring those found in chemical processing facilities. The mutants that exhibit the desired activity under those conditions will be selected for characterization and additional rounds of mutations. The project will support outreach activities for local high school and community college students that will encourage them to join the biomanufacturing workforce. Combining directed evolution with enzyme immobilization is challenging. Immobilization can cause physical changes that diminish the improvements achieved through enzyme mutation. Traditional evolution methods are low-throughput and incompatible with high-throughput screening due to detrimental effects on cell viability. The overall goal will be to establish a directed evolution workflow for surface-displayed enzymes on Bacillus subtilis spore particles. Directed evolution will be integrated with enzyme immobilization on the spore surface, leveraging the chemical resilience, retained genetic information, and proliferation capability of bacterial spores. The first step will be to develop a high-throughput fluorescence-activated cell sorting (FACS) screening workflow using engineered chemical probes to directly visualize enzymatic activity. Then, a randomized enzyme library will be generated and displayed on the spore surface. This will be followed by identification and characterization of enzyme variants with enhanced performance in harsh environments. The major developments of this research are expected to be three-fold. First, the FACS screening workflow will dramatically improve throughput for directed evolution studies. Second, the spore display strategy will further increase throughput by affording the simultaneous screening for activity in harsh conditions while immobilized. Finally, characterization of mutations that confer enhanced activity under harsh conditions could lead to design rules applicable across a wide range of enzymes with industrial relevance. 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. NSF Award ID: 2531468 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Seunghyun Sim | Institution: University of California-Irvine, IRVINE, CA | Award Amount: $343,554 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2531468 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2531468.html