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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
A Heritable Recombination system for synthetic Darwinian evolution in yeast
Dante W Romanini1, Pamela Peralta-Yahya, Vanessa Mondol
1Department of Chemistry, Columbia University, New York, NY 10027, USA.
ACS Synthetic Biology
|February 16, 2013
Summary
This study introduces Heritable Recombination, a novel system for yeast that enables controlled recombination of beneficial mutations in living cells. This synthetic biology tool enhances molecular diversity and aids in engineering biomolecules for synthetic biology applications.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Biotechnology
Background:
- Genetic recombination drives molecular diversity and evolutionary fitness.
- In vitro methods like DNA shuffling engineer biomolecules but lack in vivo control.
- Synthetic biology requires in vivo systems for controlled recombination of beneficial mutations.
Purpose of the Study:
- To develop a Heritable Recombination system for controlled in vivo mutagenesis and recombination in Saccharomyces cerevisiae.
- To optimize the system for efficient mutagenesis and successive repair of genetic markers.
- To demonstrate its application in engineering enzyme substrate specificity through combinatorial mutation crossing.
Main Methods:
- Development of a library cassette plasmid enabling inducible mutagenesis via homologous recombination.
- Utilizing repair of nonsense codons in auxotrophic markers for mutagenesis efficiency assessment.
- Employing sexual reproduction and recombination in yeast for combining beneficial mutations.
- Applying the system to alter enzyme substrate specificity through iterative mutation and selection.
Main Results:
- Achieved mutagenesis efficiencies of up to 6% in Saccharomyces cerevisiae.
- Successfully demonstrated successive repair of different auxotrophic markers over two cycles of recombination.
- Engineered a biosynthetic enzyme's substrate specificity by crossing beneficial mutations across three active site loops.
- Covered a total sequence diversity of 10^13 through combinatorial crossing.
Conclusions:
- Heritable Recombination system provides inducible mutagenesis and controlled recombination in living cells.
- The system overcomes transformation barriers for large library sizes in synthetic biology.
- It offers a powerful new tool for combinatorial crossing of beneficial mutations in cell engineering.
- This technology advances the molecular biology toolkit for next-generation synthetic biology.
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