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

Amir Aharoni, Gil Amitai, Kalia Bernath, Shlomo Magdassi, Dan S. Tawfik*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

192 Scopus citations

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 >107 serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >104 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.

Original languageAmerican English
Pages (from-to)1281-1289
Number of pages9
JournalChemistry and Biology
Volume12
Issue number12
DOIs
StatePublished - Dec 2005

Bibliographical note

Funding Information:
We are very grateful to Eitan Ariel and Ayala Sharp for their devoted assistance with the FACS. Financial support from the Israel Science Foundation (ISF) through its Bikura grant, and the Israeli Ministry of Science (IMOS), are gratefully acknowledged. D.T. is the incumbent of the Elaine Blond Career Development Chair.

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