Nature Methods 3, 609 - 614 (2006)
Published online: 21 July 2006; | doi:10.1038/nmeth899
High-throughput screening methodology for the directed evolution of glycosyltransferasesAmir Aharoni1, Karena Thieme1, Cecilia P C Chiu2, Sabrina Buchini1, Luke L Lairson1, Hongming Chen1, Natalie C J Strynadka2, Warren W Wakarchuk3 & Stephen G Withers11
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada. 2
Department of Biochemistry, Life Sciences Center, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. 3
Institute of Biological Sciences, National Research Council, Room 3157, 100 Sussex Drive, Ottawa, Ontario K1A OR6, Canada.
Correspondence should be addressed to Stephen G Withers withers@chem.ubc.ca Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of new oligosaccharides holds great potential for the development of the field of glycobiology. However, engineering of GTs by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of a new fluorescence-based high-throughput screening (HTS) methodology for the directed evolution of sialyltransferases (STs). Using this methodology, we detected the formation of sialosides in intact Escherichia coli cells by selectively trapping the fluorescently labeled transfer products in the cell and analyzing and sorting the resulting cell population using a fluorescence-activated cell sorter (FACS). We screened a library of >106 ST mutants using this methodology and found a variant with up to 400-fold higher catalytic efficiency for transfer to a variety of fluorescently labeled acceptor sugars, including a thiosugar, yielding a metabolically stable product.
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