Using enantioselective biosensors to evolve asymmetric biocatalysts

Biocatalysts are championed for their exquisite stereochemistry, but slow chromatographic separations necessary to measure enantiomeric excess can bottleneck their development. To overcome this limitation, we generate enantioselective transcription factors (eTFs) that convert enantiomer-specific analyte concentrations into programmable gene expression outputs. Using a massively parallel reporter assay, we measure dose-response curves for over 300,000 transcription factor variants in response to enantiomeric intermediates and an achiral precursor of the pharmaceutical Solifenacin. Leveraging this comprehensive dataset, we quantitatively compare the sensitivity, selectivity, and dynamic range of variants generated by random, site-saturation, and shuffling mutagenesis, enabling the isolation of eTFs with exceptional specificity. High-resolution structures further elucidate how sterics enforce enantioselectivity and charge interactions differentiate the imine precursor from amine reaction products. Finally, we use two eTFs to create a high-throughput chiral screen, which we pair with fluorescence activated cell sorting to evolve an imine reductase with inverted enantioselectivity. This method provides a rapid and scalable approach for asymmetric reaction screening, facilitating advancements in biocatalyst design for pharmaceutical manufacturing.