Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Bacterial Proteins / genetics
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Biomedical Engineering / methods*
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Directed Molecular Evolution / methods*
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Escherichia coli / genetics
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Fluorescence
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Gene Library
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Glucosyltransferases / genetics
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Glycosylation
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Glycosyltransferases / analysis*
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Glycosyltransferases / metabolism
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Mutagenesis / genetics
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Mutation / genetics
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Streptomyces antibioticus / enzymology
Substances
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Bacterial Proteins
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Glycosyltransferases
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Glucosyltransferases
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OleD protein, Streptomyces antibioticus