High-Throughput Screening of Enzyme Libraries: Thiolactonases Evolved by Fluorescence-Activated Sorting of Single Cells in Emulsion Compartments.

Citation:

Aharoni A, Amitai G, Bernath K, Magdassi S, Tawfik DS. High-Throughput Screening of Enzyme Libraries: Thiolactonases Evolved by Fluorescence-Activated Sorting of Single Cells in Emulsion Compartments. Chem. Biol. (Cambridge, MA, U. S.)Chemistry & Biology (Cambridge, MA, United States). 2005;12 (12) :1281 - 1289.

Date Published:

2005///

Abstract:

Single bacterial cells, each expressing a different library variant, were compartmentalized in aq. 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 >107 serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >104 enzyme mols., in a vol. of <10 fL (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. [on SciFinder(R)]