Genuine Directed Evolution In Test Tube (GENie)

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

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 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.

Authors 3

1. Lilin Feng Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

2. Maochao Mao Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

3. Ulrich Schwaneberg corresponding Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany