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Literature DB >> 18323806

A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution.

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.

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Year: 2008 PMID： 18323806 DOI： 10.1038/nprot.2007.538

Source DB: PubMed Journal: Nat Protoc ISSN： 1750-2799 Impact factor: 13.491

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Cited

17 in total

1. Probing the aglycon promiscuity of an engineered glycosyltransferase.

Authors: Richard W Gantt; Randal D Goff; Gavin J Williams; Jon S Thorson
Journal: Angew Chem Int Ed Engl Date: 2008 Impact factor: 15.336

2. Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization.

Authors: Gavin J Williams; Randal D Goff; Changsheng Zhang; Jon S Thorson
Journal: Chem Biol Date: 2008-04

A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution.

1. Probing the aglycon promiscuity of an engineered glycosyltransferase.

2. Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization.

3. MutS-mediated enrichment of mutated DNA produced by directed evolution in vitro.

4. Development of a universal glycosyltransferase assay amenable to high-throughput formats.

5. Recombinant E. coli prototype strains for in vivo glycorandomization.

6. Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis.

Review 7. The impact of enzyme engineering upon natural product glycodiversification.

Review 8. Opportunities for enzyme engineering in natural product biosynthesis.

9. Biochemical and structural insights of the early glycosylation steps in calicheamicin biosynthesis.

10. OleD Loki as a Catalyst for Hydroxamate Glycosylation.