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Directed evolution of a glycosynthase via chemical complementation
Hening Lin1, Haiyan Tao, Virginia W Cornish
1Department of Chemistry, Columbia University, New York, New York 10027, USA.
Journal of the American Chemical Society
|November 19, 2004
Summary
We developed Chemical Complementation, a yeast three-hybrid assay, to enhance enzyme evolution. This method successfully improved glycosynthase activity, demonstrating its utility for directed evolution of carbohydrate-active enzymes.
Area of Science:
- Biochemistry
- Enzymology
- Synthetic Biology
Background:
- Enzyme catalysis is crucial for carbohydrate synthesis.
- Directed evolution requires robust assays to screen enzyme variants.
- Chemical Complementation is a novel yeast three-hybrid assay for enzyme evolution.
Purpose of the Study:
- To apply Chemical Complementation to the directed evolution of glycosynthases.
- To enhance the activity of a glycosynthase derived from Humicola insolens Cel7B.
- To demonstrate the versatility of Chemical Complementation for new enzyme classes.
Main Methods:
- Utilized a yeast three-hybrid assay (Chemical Complementation).
- Linked glycosynthase activity of a Cel7B E197A mutant to a LEU2 reporter gene.
- Performed saturation mutagenesis at the Glu197 position and selected for improved activity.
Main Results:
- Successfully linked glycosynthase activity to cell growth via the LEU2 reporter.
- Isolated a Cel7B E197S variant with a 5-fold increase in glycosynthase activity.
- Validated Chemical Complementation as an effective platform for glycosynthase evolution.
Conclusions:
- Chemical Complementation is a versatile platform for directed evolution of glycosynthases.
- This assay facilitates the discovery of enhanced enzymes for carbohydrate synthesis.
- The approach is general and easily adaptable to other enzyme families and reactions.
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