Genuine Directed Evolution In Test Tube (GENie)

Directed evolution has long been constrained by complex screening hardware and labor-intensive workflows. Here, we report the first genuine test-tube screening platform that uses His₆-tagged peptide-functionalized magnetic beads and Fe³⁺-decorated E. coli cells to establish a phenotype-genotype linkage, thereby decoupling ultrahigh-throughput screening from specialized instrumentation and democratizing directed evolution. The platform demonstrated a screening throughput of > 10⁸ events s^-1 and an enrichment factor of up to 63-fold. Using galactose oxidase as a model, we identified variants with up to a 26-fold increase in catalytic efficiency. Extensions to D-amino acid oxidase and alcohol oxidase yielded variants with up to 5383-fold and 25-fold improvements over their respective wildtypes after a single round of screening. These results highlight the platform's capacity to rapidly engineer H₂O₂-generating oxidases and to advance AI-driven enzyme design through rapid data generation.