High-throughput screening methodology for the directed evolution of glycosyltransferases

Amir Aharoni, Karena Thieme, Cecilia P.C. Chiu, Sabrina Buchini, Luke L. Lairson, Hongming Chen, Natalie C.J. Strynadka, Warren W. Wakarchuk, Stephen G. Withers

Research output: Contribution to journalArticlepeer-review

185 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)609-614
Number of pages6
JournalNature Methods
Volume3
Issue number8
DOIs
StatePublished - 1 Aug 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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