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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
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Directed evolution of an α1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method
Yumeng Tan1, Yong Zhang1, Yunbin Han1,2
1State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Science Advances
|October 22, 2019
Summary
Researchers developed a novel screening system to improve fucosyltransferase enzymes. This directed evolution approach yielded a mutant enzyme with significantly enhanced catalytic efficiency for producing fucosylated compounds.
Area of Science:
- Biochemistry and Enzymology
- Glycobiology
- Biotechnology
Background:
- Fucosylated glycoconjugates play critical roles in numerous physiological and pathological processes.
- Economical production of fucosylated therapeutics and prebiotics is limited by low fucosyltransferase catalytic efficiency.
Purpose of the Study:
- To develop an ultrahigh-throughput screening system for improving fucosyltransferase enzymes.
- To enhance the catalytic efficiency of alpha-1,3-fucosyltransferase (α1,3-FucT) from Helicobacter pylori for industrial applications.
Main Methods:
- Established a fluorescence-activated cell sorting (FACS) system for ultrahigh-throughput screening of enzyme mutants (>10^7 mutants/hour).
- Employed directed evolution strategies over three rounds to identify improved fucosyltransferase variants.
- Utilized structural analysis and molecular dynamic simulations to understand mutation-driven functional changes.
Main Results:
- Identified a mutant M32 of α1,3-FucT with significantly improved catalytic efficiency (kcat/Km).
- Achieved 6-fold and 14-fold increases in efficiency for Lewis x and 3'-fucosyllactose synthesis, respectively.
- Structural analysis revealed an S45F mutation creating a clamp-like structure enhancing substrate binding and increased helix α5 mobility.
Conclusions:
- The developed FACS system enables rapid and efficient screening of enzyme libraries for improved fucosylation activity.
- The M32 mutant demonstrates enhanced catalytic efficiency, paving the way for cost-effective production of fucosylated compounds.
- Understanding the structural basis of enhanced activity provides insights for future enzyme engineering efforts in glycobiology.
