RUI: Development of Improved Aromatic-Metabolizing Enzymes Through the Application of Generalizable Enzyme Engineering Strategies

Many chemicals, fragrances, and drugs have molecular structures that include aromatic groups. Synthesizing these molecules is complicated and involves multiple steps, which often leads to low yields. Each step involves blocking all reactive sites on the molecule except the one targeted for a chemical reaction. Once that reaction is complete, the blocked sites must be unblocked to prepare for the next reaction. Each repetition of this cycle reduces the overall efficiency of the process. Each step also adds to the energy requirements and solvent waste generated. Using enzymes to drive reactions may reduce energy and solvent waste. This project will investigate modifying certain selected enzymes so that they can react more efficiently with molecules that contain aromatic groups. Experiments will be performed by teams of undergraduate researchers. The project will provide students with valuable experimental and analytical skills and contribute to the future U.S. biomanufacturing workforce. Previous research on enzyme catalysis has demonstrated a benefit deriving from remodeling the substrate tunnel. This strategy will be applied to three families of aromatic-metabolizing enzymes: 1) extradiol dioxygenases, 2) aromatic peroxygenases, and 3) ferulic acid decarboxylases. Variants of each of these enzyme families that have expanded reactivity for aromatic compounds will be developed using directed evolution platforms. Enzyme engineering studies will follow a “design-test-build-learn” cycle. Large variant libraries will be produced using rational engineering and will be screened for enhanced activity. AI-based protein modeling techniques will identify substitutions that might stabilize interactions between the enzyme and substrate. For each round of selection, 6-10 residues lining the substrate tunnel will be selected for mutagenesis, and variant libraries will be built through substitution of each selected residue with residues capable of establishing π-π stacking interactions with aromatic substrates (phenylalanine, tryptophan, tyrosine, histidine) and with methionine. Potential contributions of this research include A) new, sustainable catalysts with valuable applications in organic synthesis and bioremediation; and B) the demonstration of the broad practical utility of tunnel modification enzyme engineering methods. 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: 2548668 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Jordan Froese | Institution: Ball State University, MUNCIE, IN | Award Amount: $392,649 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2548668 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2548668.html