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Updated: May 22, 2026
10:50
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch.
Joshua K Michener1, Christina D Smolke
1Department of Bioengineering, 1200 E. California Blvd., MC 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Metabolic Engineering
|May 5, 2012
Summary
We developed novel in vivo biosensors using RNA switches to screen enzyme libraries in living cells. This method significantly improved enzyme activity and product selectivity for metabolic engineering applications.
Area of Science:
- Metabolic Engineering
- Synthetic Biology
- Biotechnology
Background:
- Metabolic engineering utilizes renewable resources for chemical production.
- High activity of heterologous enzymes is crucial but challenging to achieve in whole cells.
- Directed evolution is effective but difficult to apply in vivo.
Purpose of the Study:
- To develop generalizable in vivo biosensors for screening enzyme libraries.
- To link metabolite concentrations to reporter gene expression in living cells.
- To overcome limitations of in vitro directed evolution for whole-cell biocatalysis.
Main Methods:
- Engineered RNA switches were designed to create biosensors.
- Biosensors linked metabolite levels to Green Fluorescent Protein (GFP) expression.
- High-throughput screening was performed using fluorescence and Fluorescence-Activated Cell Sorting (FACS).
Main Results:
- Iterative screening of a caffeine demethylase library yielded beneficial mutations.
- Enzyme activity in vivo was increased by 33-fold.
- Product selectivity was enhanced by 22-fold.
Conclusions:
- RNA-based biosensors provide a powerful tool for in vivo enzyme screening.
- This technique is adaptable to various enzymes and metabolic pathways via aptamer selection.
- The developed method accelerates the optimization of biocatalysts for chemical production.
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