title: High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments.
abstract: 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 enzyme-coding 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 femtoliter (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.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Amir
authors.initials: A
authors.lastname: Aharoni
authors.forename: Gil
authors.initials: G
authors.lastname: Amitai
authors.forename: Kalia
authors.initials: K
authors.lastname: Bernath
authors.forename: Shlomo
authors.initials: S
authors.lastname: Magdassi
authors.forename: Dan S
authors.initials: DS
authors.lastname: Tawfik
mesh_terms: D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods; D004655:Emulsions; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D005434:Flow Cytometry / Q000379:methods*; D015723:Gene Library; D009154:Mutation; D015202:Protein Engineering / Q000379:methods
chemical_list: D004655:Emulsions; D043303:Aryldialkylphosphatase