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A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of
Gheorghita Menghiu1,2, Vasile Ostafe2, Radivoje Prodanović3
1Institute for Biology VII, Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
International Journal of Molecular Sciences
|April 3, 2021
Summary
Researchers developed a novel high-throughput screening system using fluorescence-activated cell sorting to enhance chitinase enzymes. This method identified improved chitinase variants with up to twofold higher activity, advancing sustainable chitin degradation.
Area of Science:
- Biotechnology
- Enzymology
- Molecular Evolution
Background:
- Chitinases degrade chitin, a biopolymer with applications in various industries.
- Current chitinase enzymes are slow-acting, limiting their biotechnological use.
- Enzymatic chitin degradation offers a sustainable alternative to chemical methods.
Purpose of the Study:
- To develop a high-throughput screening system for chitinase activity.
- To identify improved chitinase variants with enhanced catalytic efficiency.
- To address limitations in current chitinase applications and environmental chitin waste.
Main Methods:
- Cloning and expression of *Bacillus licheniformis* chitinase A (ChiA) in *Escherichia coli*.
- Generation of a mutant library using error-prone PCR.
- Development of a fluorescence-activated cell sorting (FACS) based screening assay using a fluorogenic substrate.
- Optimization of the assay to prevent product-induced cross-talk and mitigate host cell toxicity.
Main Results:
- Successful development of a robust FACS-based screening system for chitinase activity.
- Identification of 12 improved ChiA variants with 2-8 mutations each.
- Achieved up to a twofold increase in enzyme activity compared to wild-type ChiA.
- Demonstrated the utility of the screening system for directed evolution of hydrolytic enzymes.
Conclusions:
- The developed FACS screening system is effective for identifying enhanced chitinases.
- Molecular evolution can significantly improve chitinase performance for biotechnological applications.
- This approach offers a sustainable solution for chitin waste management and chitosan production.
