Genuine Directed Evolution In Test Tube (GENie)

Feng, L.; Mao, M.; Schwaneberg, U.

doi:10.64898/2026.05.04.722721

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bioRxiv · 10.64898/2026.05.04.722721

Genuine Directed Evolution In Test Tube (GENie)

Feng, L.·Mao, M.·Schwaneberg, U.

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Abstract

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 platforms capacity to rapidly engineer H2O2-generating oxidases and to advance AI-driven enzyme design through rapid data generation.

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Feng, L., Mao, M., Schwaneberg, U.. 2026-05-07. Genuine Directed Evolution In Test Tube (GENie). https://doi.org/10.64898/2026.05.04.722721

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