RUI: development of improved aromatic-metabolizing enzymes through the application of generalizable enzyme engineering strategies

The National Science Foundation Division of Chemical, Bioengineering, Environmental, and Transport Systems awarded Ball State University $392,649 on May 1, 2026, to develop improved aromatic-metabolizing enzymes through enzyme engineering strategies under the Engineering program (CFDA 47.041).

The project will engineer three families of aromatic-metabolizing enzymes—extradiol dioxygenases, aromatic peroxygenases, and ferulic acid decarboxylases—to react more efficiently with molecules containing aromatic groups. The work applies directed evolution platforms and artificial intelligence–based protein modeling to identify enzyme variants with expanded reactivity. Each round of engineering will follow a design-test-build-learn cycle, with mutagenesis targeting 6–10 residues lining the substrate tunnel. The goal is to reduce energy and solvent waste in chemical synthesis of fragrances, drugs, and other aromatic-containing molecules by replacing multi-step chemical synthesis with enzyme-driven reactions.

Undergraduate research teams at Ball State will conduct the experiments, providing students with experimental and analytical skills training as part of the project deliverable. Performance occurs in Muncie, Indiana, with a period of performance from May 1, 2026, through April 30, 2029. This is a Research in Undergraduate Institutions (RUI) award, classified as a Project Grant.

Abstract

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 ?-? 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.- subawards are not planned for this award.