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Related Concept Videos

Genetic Screens02:46

Genetic Screens

Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing  genetic diseases and help them with early intervention, targeted therapy, and reproductive options.
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which result in visible changes...

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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
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A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution.

Gavin J Williams1, Jon S Thorson

  • 1Laboratory 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.

Nature Protocols
|March 8, 2008
PubMed
Summary

This study presents a method using high-throughput screening and mutagenesis to engineer glycosyltransferase (GT) enzymes. The approach aims to improve enzyme function for secondary metabolite production and can be applied to similar enzyme families.

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Area of Science:

  • Biochemistry
  • Enzyme Engineering
  • Molecular Biology

Background:

  • Secondary metabolite glycosyltransferases (GTs) are crucial for producing diverse natural products.
  • Modifying GTs can enhance or alter their catalytic properties, impacting metabolite profiles.
  • Directed evolution offers a powerful strategy for enzyme optimization.

Purpose of the Study:

  • To detail a protocol for engineering secondary metabolite GTs.
  • To improve GT enzyme proficiency and/or promiscuity using directed evolution.
  • To provide a template for engineering other GT-B superfamily members.

Main Methods:

  • Application of a high-throughput fluorescence-based screen.
  • Utilizing error-prone PCR and saturation mutagenesis for enzyme variant generation.
  • Employing directed evolution to select for altered GT activity.

Main Results:

  • Successfully developed a protocol for high-throughput screening of GT variants.
  • Demonstrated the ability to alter GT enzyme proficiency and promiscuity.
  • Established a framework applicable to engineering related enzymes.

Conclusions:

  • The described protocol enables efficient engineering of secondary metabolite GTs.
  • This directed evolution approach is effective for modifying enzyme function.
  • The methodology serves as a template for broader applications within the GT-B superfamily.