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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus
S Camarero1, I Pardo, A I Cañas
1Department of Biocatalysis, Institute of Catalysis, Madrid, Spain.
Applied and Environmental Microbiology
|January 3, 2012
Summary
Directed evolution of Pycnoporus cinnabarinus laccase (PcL) in yeast significantly enhanced its activity by 8,000-fold. This breakthrough enables improved biocatalysis through engineered enzymes with better secretion and catalytic efficiency.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Environmental Biocatalysis
Background:
- Pycnoporus cinnabarinus laccase (PcL) shows promise for environmental biocatalysis due to its high redox potential.
- Practical applications of PcL are hindered by limitations in directed evolution platforms for improving enzyme characteristics.
Purpose of the Study:
- To develop a directed evolution platform for PcL in Saccharomyces cerevisiae.
- To engineer PcL for enhanced activity, secretion, and broader applicability in biocatalysis.
Main Methods:
- Constructed a PcL fusion gene with an α-factor preproleader, replacing the native signal peptide.
- Performed six rounds of directed evolution using a multiscreening assay with natural and synthetic redox mediators at neutral pH.
- Utilized Saccharomyces cerevisiae for functional expression and Aspergillus niger for high-level secretion.
Main Results:
- Achieved an 8,000-fold increase in total laccase activity.
- Enhanced secretion levels by 40-fold due to the evolved α-factor preproleader.
- Increased catalytic efficiency (kcat) by 13.7-fold through mutations in the mature laccase, while retaining thermostability and substrate specificity.
Conclusions:
- Successfully established a directed evolution platform for PcL in yeast, enabling significant enzyme improvement.
- Demonstrated the potential of the evolved PcL variants for high-level secretion in Aspergillus niger.
- Identified key mutations that enhance laccase performance and provide insights into substrate oxidation mechanisms.
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