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Article Abstract
Purpose: High-throughput screening methods for cytochrome P450 enzymes (P450s), such as colorimetric, mass spectrometric, and fluorescence-based assays, often face limitations in throughput, real-time monitoring, and versatility.
Methods: To address these challenges, we developed a novel biosensor leveraging glucose-6-phosphate dehydrogenase and Bimolecular Fluorescence Complementation for real-time monitoring of intracellular NADP levels, enabling P450 activity detection. The sensor was applied to monitor P450 activity by tracking intracellular NADP dynamics, as P450s catalyze diverse substrate reactions and convert NADPH to NADP via their electron transport system. To enhance detection precision, intracellular NADP synthesis was reduced by knocking down NADPH-dependent aldehyde reductase (YqhD), minimizing background fluorescence interference.
Results: The sensor exhibited a linear NADP detection range of 1 μM to 10 mM, suitable for P450 assays. The sensor's performance was validated by comparing P450 activities in engineered strains with traditional gas chromatography.
Conclusion: The developed biosensor demonstrates its potential as a robust, real-time screening tool for P450 enzyme studies.
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