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Updated: Jun 7, 2026
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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Ultrahigh-throughput screening system for directed glucose oxidase evolution in yeast cells
Radivoje Prodanovic1, Raluca Ostafe, Andreea Scacioc
1RWTH Aachen University, Lehrstuhl für Biotechnologie, Worringer Weg 1, 52056 Aachen, Germany.
Combinatorial Chemistry & High Throughput Screening
|October 21, 2010
Summary
A new compartmentalized tyramide labeling system (CoaTi) enables ultrahigh-throughput screening of Glucose oxidase (GOx) variants. This method identified a GOx variant with significantly enhanced catalytic efficiency.
Area of Science:
- Biotechnology
- Enzyme Engineering
- High-Throughput Screening
Background:
- Developing efficient screening methods is crucial for enzyme engineering.
- Glucose oxidase (GOx) is an important industrial enzyme with potential for improvement.
Purpose of the Study:
- To develop an ultrahigh-throughput screening system for Glucose oxidase (GOx) variants.
- To identify novel GOx variants with improved enzymatic properties.
Main Methods:
- A compartmentalized tyramide labeling system (CoaTi) was developed using yeast cells in microdroplets.
- Flow cytometry was employed for sorting cells based on peroxidase activity.
- An error-prone PCR mutant library of GOx was screened for improved activity.
Main Results:
- The CoaTi system successfully screened a library of ~10^5 GOx variants.
- A variant, Mut12, with 5 mutations exhibited a 2.7-fold increase in catalytic rate (kcat) and a decreased Michaelis constant (Km).
- Mut12 showed a 1.8-fold increased kcat compared to the parent B11 GOx.
Conclusions:
- The CoaTi system is effective for ultrahigh-throughput screening of enzyme variants.
- Engineered GOx variants can exhibit significantly enhanced catalytic performance.
- This approach facilitates the discovery of improved enzymes for industrial applications.
