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Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based
R G Rosenthal1,2, X Diana Zhang1, K Ilić Đurđić1,3,4
1School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
Angewandte Chemie (International Ed. in English)
|April 5, 2023
Summary
This study introduces a continuous evolution platform for enzyme optimization, significantly reducing labor and capital costs. The new method enhances enzyme catalytic efficiency, turning waste products into valuable feedstocks.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Synthetic Biology
Background:
- Enzymes are crucial catalysts for drug development and industrial processes, but often require optimization.
- Directed evolution is a common method for enzyme optimization but is labor- and capital-intensive due to multiple molecular biology steps and limited screening throughput.
Purpose of the Study:
- To develop an effective and broadly applicable continuous evolution platform for ultrahigh-throughput enzyme evolution.
- To enable controlled exploration of enzyme fitness landscapes by directly measuring enzymatic activity.
Main Methods:
- A drop-based microfluidics platform was utilized for continuous evolution.
- The platform integrates cell growth, mutagenesis (using nCas9 chimera and mutagenesis polymerase), and screening with minimal human intervention.
- In vivo gene diversification was achieved using sgRNAs tiled along the target gene.
Main Results:
- The platform was successfully used to evolve alditol oxidase, altering its substrate specificity towards glycerol.
- A variant with a 10.5-fold increase in catalytic efficiency was identified.
- This demonstrates the platform's ability to convert waste products into valuable feedstocks.
Conclusions:
- The developed continuous evolution platform offers an effective and broadly applicable solution for enzyme engineering at ultrahigh throughput.
- This technology significantly reduces the labor and capital investment required for enzyme optimization.
- The platform facilitates the development of novel biocatalysts with tailored substrate specificities, with potential applications in waste valorization and green chemistry.
