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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 mandelate racemase by a novel high-throughput screening method
Chengcheng Yang1, Lidan Ye1,2, Jiali Gu3
1Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
Applied Microbiology and Biotechnology
|August 26, 2016
Summary
Directed evolution enhanced mandelate racemase (MR) activity towards (S)-2-chloromandelic acid ((S)-2-CMA). A novel high-throughput screening method enabled the development of a triple mutant MRDE1 with 3.5-fold greater catalytic efficiency.
Area of Science:
- Enzyme engineering
- Biocatalysis
- Medicinal chemistry
Background:
- Optically pure intermediates like (R)-o-chloromandelate are crucial for synthesizing (S)-clopidogrel.
- Current methods for sequential hydrolysis and racemization are limited by low mandelate racemase (MR) activity on (S)-o-chloromandelic acid ((S)-2-CMA).
Purpose of the Study:
- To improve the catalytic performance of mandelate racemase (MR) towards (S)-2-chloromandelic acid ((S)-2-CMA) using directed evolution.
- To develop an enantioselective oxidation system for high-throughput screening (HTS) of MR libraries.
Main Methods:
- Development of an enantioselective oxidation system for high-throughput screening (HTS).
- Application of directed evolution to engineer mandelate racemase (MR).
- Kinetic analysis and molecular simulations to understand enzyme-substrate interactions.
Main Results:
- A triple mutant (V22I/V29I/Y54F, designated MRDE1) exhibited a 3.5-fold increase in relative activity compared to wild-type MR.
- Enhanced catalytic efficiency was primarily attributed to an elevated k cat.
- Molecular simulations suggested improved substrate binding and product release due to reduced steric hindrance and increased hydrophobicity in the active site.
Conclusions:
- The developed HTS method and successful directed evolution of MR provide a viable strategy for racemase engineering.
- This work offers a pathway for enhancing racemase performance on non-natural substrates, potentially inspiring future enzyme evolution efforts.
