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Growth-Based, High-Throughput Selection for NADH Preference in an Oxygen-Dependent Biocatalyst
Sarah Maxel1, Samer Saleh1, Edward King2
1Departments of Chemical and Biomolecular Engineering, University of California, Irvine, California 92697, United States.
ACS Synthetic Biology
|September 1, 2021
Summary
Researchers engineered cyclohexanone monooxygenases (CHMO) to use NADH instead of NADPH, overcoming stability and specificity limitations. This advance utilized a novel high-throughput selection platform for industrial enzyme applications.
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Synthetic Biology
Background:
- Cyclohexanone monooxygenases (CHMO) are valuable enzymes for cyclic ketone oxidation but suffer from poor stability and strict NADPH cofactor dependence.
- Engineering CHMO is challenging due to enzyme sensitivity to conformational dynamics and unpredictable long-range interactions.
Purpose of the Study:
- To develop a high-throughput screening platform for oxygenase evolution using NADH redox balance in *Escherichia coli*.
- To engineer CHMO variants with altered cofactor specificity, enabling the use of the less expensive NADH cofactor.
Main Methods:
- Developed an aerobic, high-throughput growth selection platform in *Escherichia coli* for oxygenase evolution.
- Employed semirational design to identify CHMO DTNP, a variant with significantly altered cofactor specificity.
- Utilized random mutagenesis on CHMO DTNP to further enhance cofactor specificity.
Main Results:
- Identified CHMO DTNP (S208D-K326T-K349N-L143P) with a ~1200-fold relative switch in cofactor specificity from NADPH to NADH.
- Molecular modeling indicated that CHMO DTNP's activity results from cooperative fine-tuning of cofactor contacts.
- Further evolution yielded CHMO DTNPY, exhibiting a ~2900-fold relative specificity switch due to an additional H163Y mutation.
Conclusions:
- Demonstrated the effectiveness of a high-throughput NADH-dependent selection platform for engineering oxygenases.
- Highlighted the limitations of static models and rational design, emphasizing the need for selection-based evolution.
- Developed novel CHMO variants with enhanced cofactor specificity, paving the way for industrial applications.
Keywords:
Baeyer−Villiger monooxygenasecofactor specificitycyclohexanone monooxygenasedirected evolutionhigh-throughput selectionredox balanceMore Related Videos
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