Fluorescence activated cell sorting as a general ultra-high-throughput screening method for directed evolution of glycosyltransferases

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DOIResolve DOI: http://doi.org/10.1021/ja104167y
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TypeArticle
Journal titleJournal of the American Chemical Society
ISSN1520-5126
0002-7863
Volume132
Issue30
Pages1057010577; # of pages: 8
AbstractGlycosyltransferases (GTs) offer very attractive approaches to the synthesis of complex oligosaccharides. However, the limited number of available GTs, together with their instability and strict substrate specificity, have severely hampered the broad application of these enzymes. Previous attempts to broaden the range of substrate scope and to increase the activity of GTs via protein engineering have met with limited success, partially because of the lack of effective high-throughput screening methods. Recently, we reported an ultra-high-throughput screening method for sialyltransferases based on fluorescence-activated cell sorting (Aharoni et al. Nat. Methods 2006, 3, 609-614). Here, we considerably improve this method via the introduction of a two-color screening protocol to minimize the probability of false positive mutants and demonstrate its generality through directed evolution of a neutral sugar transferase, ?-1,3-galactosyltransferase CgtB. A variant with broader substrate tolerance than the wildtype enzyme and 300-fold higher activity was identified rapidly from a library of >107 CgtB mutants. Importantly, the variant effected much more efficient synthesis of GM1a and asialo GM1 oligosaccharides, the building blocks of important therapeutic glycosphingolipids, than did the parent enzyme. This work not only establishes a new methodology for the directed evolution of galactosyltransferases, but also suggests a powerful strategy for the screening of almost all GT activities, thereby facilitating the engineering of glycosyltransferases.
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedYes
NPARC number17401015
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Record identifierac94e898-69f1-48e3-91dd-3b45d051d4bf
Record created2011-03-25
Record modified2016-05-09
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