Directed Evolution of Glycosyltransferases by a Single-Cell Ultrahigh-Throughput FACS-Based Screening Method

Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of complex oligosaccharides has been of great scientific and industrial interest. Here we describe an ultra-high-throughput fluorescence activated cell sorting (FACS) method for the directed evolution of GTs, at the single cell level. This assay relies on the exquisite substrate specificity of lactose permeases (LacY) that are located in the cell membrane, which distinguish selectively the fluorescent glycosylated products from the unreacted substrates. The method described here allows facile screening 10⁶-10⁷ mutants per hour. We demonstrate the application of this technique in the screening of libraries of α1,3-fucosyltransferase.