Using enantioselective biosensors to evolve asymmetric biocatalysts

Simon d’Oelsnitz, Wantae Kim, Nicole N Zhao, Haley A. Hardtke, Svetlana P. Ikonomova, Nina Alperovich, Olga Vasilyeva, Michael J. James, Eric Zigon, Michael Cory, Connor Johnson, Andrew D. Ellington, Quincey Justman, Michael Springer, Yan Zhang, Pamela A. Silver, David Ross

Biocatalysts are prized for their enantioselectivity, but slow chromatographic separations required to measure enantiomeric excess bottleneck their development. To overcome this limitation, we evolve enantioselective transcription factors (eTFs) that convert enzyme-catalyzed enantiomer concentrations into programmable gene expression outputs, focusing on imine reductases. Here, using a massively parallel reporter assay, we measure dose–response curves for over 300,000 transcription factor variants in response to an imine precursor and chiral amine products. We quantify the sensitivity, selectivity and dynamic range across variants generated by random, site-saturation and shuffling mutagenesis, isolating variants with exceptional specificity. High-resolution structures of evolved eTFs elucidate how steric effects enforce enantioselectivity, while charge interactions distinguish the imine from the amines. Using two eTFs, we create an ultrahigh-throughput chiral screen to evolve an imine reductase with inverted enantioselectivity. To support generalizability and speed, we design a genetic circuit that enables TF generation within weeks. Our methods enable rapid measurement of asymmetric reactions, supporting innovation in chemical manufacturing. Here d’Oelsnitz et al. generate enantioselective transcription factors that convert chiral reaction products into fluorescent signals, enabling the rapid evolution of asymmetric enzymes with inverted selectivity.