High-throughput screening of enzyme libraries: Thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments

Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzymecoding genes. We demonstrate the directed evolution of new enzyme variants by screening > 10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying > 10(4) enzyme molecules, in a volume of < 10 ferntoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.