Directed evolution has been remarkably successful in identifying enzyme variants with new or improved properties, such as altered substrate scope or novel reactivity. Genetically encodable biosensors (GEBs), which convert the concentration of a small molecule ligand into an easily detectable output signal, have seen increasing application to enzyme directed evolution in the last decade. GEBs enable the use of high-throughput methods to assess enzyme activity of very large libraries, which can accelerate the search for variants with desirable activity. Here, we review different classes of GEBs and their properties in the context of enzyme evolution, how GEBs have been integrated into directed evolution workflows, and recent examples of enzyme evolution efforts utilizing GEBs. Finally, we discuss the advantages, challenges, and opportunities for using GEBs in the directed evolution of enzymes.
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