High-throughput screening methodology for the directed evolution of glycosyltransferases

被引:164
作者
Aharoni, Amir
Thieme, Karena
Chiu, Cecilia P. C.
Buchini, Sabrina
Lairson, Luke L.
Chen, Hongming
Strynadka, Natalie C. J.
Wakarchuk, Warren W.
Withers, Stephen G.
机构
[1] Univ British Columbia, Dept Chem, Vancouver, BC V6T 1Z1, Canada
[2] Life Sci Ctr, Dept Biochem, Vancouver, BC V6T 1Z3, Canada
[3] Natl Res Council Canada, Inst Biol Sci, Ottawa, ON K1A 0R6, Canada
关键词
D O I
10.1038/NMETH899
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:609 / 614
页数:6
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