Multiplexed Ultrahigh-Throughput Evolution for Esterase Selectivity across a Broad Substrate Scope

Abstract Directed evolution of enzymes is generally performed toward a specific substrate, which predictably improves enzyme activity toward the probe substrate. However, it is difficult to predict whether evolved enzymes will be selective or promiscuous toward other substrates. Multiplexed directed evolution can identify enzyme variants with selectivity/promiscuity across multiple substrates, but is typically performed using plate-based screening assays that are limited to testing a few hundred mutants. Combining substrate multiplexing with ultrahigh-throughput evolution technologies is a promising avenue for deeper exploration of the mutational landscape, but it has not been investigated whether enzymes evolved using multiplexed ultrahigh-throughput methods exhibit selectivity/promiscuity toward a broader scope of small-molecule substrates beyond the probes used during evolution. Here, we performed multiplexed ultrahigh-throughput evolution of Bacillus subtilis (BS2) esterase, merging yeast surface display with masked acid chloride probes, to identify a panel of mutants with improved selectivity toward the phenylcyclopropyl (pCP) ester protecting group relative to not only the probe substrate, but also a broad panel of 90 additional esters. Additionally, we applied the platform to identify a promiscuous mutant with improved activity across a broad substrate scope. Finally, we applied the highly pCP-selective BS2 variant, together with a selective variant for the methylcyclopropyl (mCP) ester protecting group, to demonstrate proximity labeling in mammalian cells with two orthogonal enzyme/probe systems, opening new opportunities in multiplexed proximity labeling. Overall, this study presents an efficacious method to identify enzyme variants with desired selectivity profiles that may be extended to multiple applications in chemical biology and synthesis.