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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
Published on: August 8, 2016
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Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol
Christian Rapp1, Simone Pival-Marko1,2, Erika Tassano3
1Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, 8010, Graz, Austria.
BMC Biotechnology
|October 12, 2021
Summary
Engineered xylose reductase (CtXR D51A) enables high-titer production of (S)-2-phenylpropanol, overcoming limitations of previous biocatalytic methods. This optimized whole-cell system achieves 115 g/L with 93.1% enantiomeric excess, offering a competitive alternative to chemical synthesis.
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Organic Synthesis
Background:
- Previous biocatalytic routes for enantiopure 2-phenylpropanol using oxidoreductases were limited to 80 mM product titers.
- Enzyme deactivation due to aldehyde substrate adduct formation with reductase amino acid residues was the primary limitation.
Purpose of the Study:
- To develop a highly efficient and stable biocatalytic system for producing enantiopure (S)-2-phenylpropanol.
- To overcome enzyme deactivation issues encountered in prior oxidoreductase-catalyzed reactions.
Main Methods:
- Engineered a single point mutant of Candida tenuis xylose reductase (CtXR D51A) for enhanced activity towards (S)-2-phenylpropanal.
- Utilized a whole-cell biocatalyst co-expressing the engineered reductase and yeast formate dehydrogenase for NADH recycling.
- Optimized substrate-to-catalyst ratio (3.4 g substrate/g cell-dry-weight) and catalyst loading (40 g cell-dry-weight) for maximal yield and enantioselectivity.
Main Results:
- The CtXR D51A mutant exhibited high catalytic efficiency (43·10³ s⁻¹ M⁻¹) and enantioselectivity for (S)-2-phenylpropanal.
- A whole-cell system significantly stabilized the reductase, enabling the conversion of 1 M racemic 2-phenylpropanal.
- Achieved a product titer of 843 mM (115 g/L) (S)-2-phenylpropanol with 93.1% enantiomeric excess.
Conclusions:
- The developed bioreduction route is a viable and competitive alternative to current industrial profenol production methods relying on hydrolases.
- This engineered biocatalyst system demonstrates superior performance compared to previously reported oxidoreductase-based approaches.
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