Emerging biosensor and assay-enabled high-throughput screening solutions for enzyme and strain engineering.

High-throughput screening (HTS) platforms and automated biofoundries have enabled large-scale experimentation in enzyme and microbial strain engineering. Central to HTS are biosensors and assays, which translate biochemical activities into measurable signals, enabling rapid evaluation of cellular and enzymatic performance. Yet despite advancements in high-throughput infrastructure, the limited availability of robust biosensors or assays and the difficulty of integrating them with HTS, particularly with ultra-HTS, remains a major bottleneck. This review highlights recent progress and challenges in applying biosensors- and assays-enabled HTS for enzyme and strain libraries. We discuss strategies for integrating diverse biosensor types, including transcription factors, G protein-coupled receptors, aptamers, fluorogenic RNAs, riboswitches, and colorimetric assays, with HTS to detect a broad range of metabolites and products. We also explore how biosensor-enabled HTS facilitates data generation for machine learning-guided biocatalyst engineering. Collectively, these advances accelerate biocatalyst discovery and drive the next generation of sustainable biomanufacturing.