title: High-throughput screening methodology for the directed evolution of glycosyltransferases.
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 >10(6) 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.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Amir
authors.initials: A
authors.lastname: Aharoni
authors.forename: Karena
authors.initials: K
authors.lastname: Thieme
authors.forename: Cecilia P C
authors.initials: CP
authors.lastname: Chiu
authors.forename: Sabrina
authors.initials: S
authors.lastname: Buchini
authors.forename: Luke L
authors.initials: LL
authors.lastname: Lairson
authors.forename: Hongming
authors.initials: H
authors.lastname: Chen
authors.forename: Natalie C J
authors.initials: NC
authors.lastname: Strynadka
authors.forename: Warren W
authors.initials: WW
authors.lastname: Wakarchuk
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001681:Biological Assay / Q000379:methods; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000502:physiology*; D019143:Evolution, Molecular; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D016695:Glycosyltransferases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D016695:Glycosyltransferases