A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution.
This protocol details the application of a high-throughput fluorescence-based screen, in conjunction with error-prone PCR/saturation mutagenesis, for altering the proficiency and/or promiscuity of a secondary metabolite glycosyltransferase (GT) via directed evolution. Given the structural and mechanistic similarities among secondary metabolite-associated GTs, this approach may provide a template for engineering other members of the GT-B superfamily.
Williams
Gavin J
GJ
Laboratory for Biosynthetic Chemistry, Pharmaceutical Sciences Division, School of Pharmacy, National Cooperative Drug Discovery Program, University of Wisconsin-Madison, 777 Highland Avenue, Madison, Wisconsin 53705, USA.
Thorson
Jon S
JS
D001426:Bacterial Proteins / Q000235:genetics; D001698:Biomedical Engineering / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D015723:Gene Library; D005964:Glucosyltransferases / Q000235:genetics; D006031:Glycosylation; D016695:Glycosyltransferases / Q000032:analysis* / Q000378:metabolism; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D013303:Streptomyces antibioticus / Q000201:enzymology
D001426:Bacterial Proteins; D016695:Glycosyltransferases; D005964:Glucosyltransferases; C519503:OleD protein, Streptomyces antibioticus