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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
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Whole-Cell-Catalyzed Multiple Regio- and Stereoselective Functionalizations in Cascade Reactions Enabled by Directed
Aitao Li1,2, Adriana Ilie1,2, Zhoutong Sun1,2
1Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany.
Angewandte Chemie (International Ed. in English)
|August 31, 2016
Summary
Directed evolution created engineered E. coli whole cells for novel biocatalytic cascade reactions. This enables efficient synthesis of valuable chiral products like cyclohexane-1,2-diol from simple starting materials.
Area of Science:
- Biocatalysis and metabolic engineering
- Synthetic chemistry
- Enzyme engineering
Background:
- One-pot biocatalytic cascade reactions offer efficient synthesis of chiral products.
- Current limitations include substrate specificity of wild-type enzymes.
- Developing novel biocatalytic routes is crucial for sustainable chemistry.
Purpose of the Study:
- To engineer E. coli whole cells for novel regio- and stereoselective biocatalytic cascade reactions.
- To overcome substrate limitations of wild-type enzymes using directed evolution.
- To demonstrate the synthesis of valuable chiral diols and hydroxyketones.
Main Methods:
- Directed evolution of E. coli whole cells.
- Utilizing P450-BM3 and alcohol dehydrogenases (ADHs) as biocatalysts.
- Implementing two-, three-, and four-step cascade reactions in one-pot processes.
Main Results:
- Engineered E. coli successfully performed cascade reactions starting from cyclohexane, cyclohexanol, or cyclohexanone.
- Achieved stereoselective synthesis of (R,R)-, (S,S)-, and meso-cyclohexane-1,2-diol.
- Demonstrated one-pot conversion of cyclohexane to (R)- or (S)-2-hydroxycyclohexanone.
Conclusions:
- Directed evolution is a powerful tool for creating bespoke biocatalytic systems.
- Engineered whole cells enable complex cascade reactions not feasible with native enzymes.
- This approach expands the scope of biocatalysis for producing valuable chiral compounds.
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