Directed Evolution of Enzymes for Bioorthogonal Chemistry Using Acid Chloride Proximity Labeling

Combining bioorthogonal protecting groups with localized catalysts that can unmask them is a powerful approach to spatially and temporally modulate molecular activity. Enzymes are appealing catalysts in this context because they are genetically targetable, but enzymes are not always available to unmask a protecting group of interest. Here, we report a platform for ultrahigh-throughput enzyme evolution by combining yeast surface display with masked acylating probes, which selectively label yeast cells based on target biocatalytic activity. We introduce the phenylcyclopropyl (pCP) ester protecting group, which has improved bioorthogonality compared to existing ester protecting groups, and use our platform to evolve BS2 esterase for enhanced pCP unmasking. Evolved BS2 mutants are up to 232-fold more active toward the pCP group. Taking advantage of the enhanced bioorthogonality of the pCP group, we applied a pCP probe together with evolved BS2 to perform spatially resolved RNA tagging with high spatial specificity, including in mammalian cell lines with high endogenous esterase activity. Overall, this work delivers a new bioorthogonal protecting group and engineered enzymes capable of unmasking it, and more broadly, it provides a platform to rapidly engineer enzymes for protecting group removal, opening opportunities in imaging, proximity tagging, induced cell signaling, and therapeutics.