Antibodies and other binding proteins are indispensable tools for molecular recognition, but these protein- based reagents lack key chemical features found in small molecules that mediate enzyme inhibition, covalent target engagement, and other function-disrupting bioactivities. I hypothesize that “chemically expanding” antibodies using genetic code manipulation offers opportunities to discover unique, function-disrupting hybrids that cannot be accessed using conventional proteins or small molecules. To realize the full potential of this approach, my group is integrating noncanonical amino acid (ncAA) incorporation with yeast display. We have previously A) characterized and improved genetic code expansion in yeast to broaden access to ncAAs during yeast display; and B) discovered potent, “chemically expanded” enzyme inhibitors in high-throughput screens. Building off of these initial successes, we propose to pursue the following three directions: Direction 1: “Harmonize” yeast ncAA incorporation systems with tools available in other cell types to expand antibody chemical diversity. Most tools for genetic code expansion are incompatible with the yeast translation apparatus or are otherwise poorly active in yeast. We propose to adapt a versatile existing orthogonal translation system (OTS) used in E. coli and mammalian cells for use in yeast. Our engineering approach leverages our OTS engineering expertise and has the potential to dramatically but efficiently expand the chemistries available for genetic code expansion in yeast. Direction 2: Elucidate combinations of molecular and genetic/genomic engineering strategies that improve ncAA incorporation in yeast. Our prior work has identified several approaches to enhancing ncAA incorporation in yeast, including unprecedented strain engineering and translation apparatus engineering. We propose to investigate which combinations of strategies yield additive or synergistic improvements to ncAA incorporation. These are fundamentally important investigations of genetic code expansion systems that, to our knowledge, have never been studied in any organism. Direction 3: Illuminate the interplay between chemical functionality and synthetic antibody diversity during enzyme inhibitor discovery. NcAAs enable presentation of chemistries that inhibit enzymes via rapid bioorthogonal conjugations and directly through ncAA side chain chemistries. We propose to use first-generation and newly designed second-generation ncAA-containing antibody libraries in combination with metalloproteinase and protein tyrosine phosphatase targets to understand how to best leverage both conjugates and directly encoded chemistries during inhibitor discovery. Our uniquely positioned research program will reveal crucial principles of protein biosynthesis and enzyme inhibitor discovery while establishing powerful tools (genetic code expansion systems and enzyme inhibitors) for understanding and treating human disease.