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 His 6 -tagged peptide functionalized magnetic beads and Fe 3+ -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 > 10 8 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 H 2 O 2 -generating oxidases and to advance AI-driven enzyme design through rapid data generation.