High-Throughput Measurement of Small-Molecule Enantiopurity by Using Flow Cytometry

Fluorescence-activated cell sorting (FACS) offers a powerful approach to high-throughput library screening in directed evolution experiments. However, FACS is rarely used in the evolution of stereoselective enzymes, due to the difficulty of designing fluorescence-based assays for measuring enantiopurity. Here, we describe a new FACS-based enantiopurity analysis approach that overcomes these limitations by using enantiomeric DNA biosensors labeled with orthogonal fluorophores. By co-encapsulating the biosensors with a mixture of target enantiomers in microfluidic droplets, we could demonstrate the use of FACS to differentiate between droplets having various levels of target enantiopurity. We envision the utility of this method for high-throughput screening of enantiopurity in the directed evolution of stereoselective enzymes, thereby facilitating the discovery of new asymmetric biocatalysts for the synthesis of pharmaceuticals and other high-value chemicals.