In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

Area of Science:

• Biocatalysis and Enzyme Engineering
• Protein Engineering
• Industrial Biotechnology

Background:

• Cyclohexanone monooxygenase (CHMO) exhibits broad substrate specificity for biooxidizing cyclic ketones into esters and lactones with high stereospecificity.
• The industrial application of CHMO is limited by its poor thermostability and challenges in protein engineering for enhanced stability.
• Existing high-throughput screening methods for enzyme engineering are limited, hindering efforts to improve CHMO's thermal resilience.

Purpose of the Study:

• To develop and apply a high-throughput growth selection platform in Escherichia coli for discovering thermostability-enhancing mutations in CHMO.
• To engineer a more robust and industrially viable CHMO variant through directed evolution and rational design.
• To demonstrate the efficacy of a novel in vivo screening system for rapid protein engineering of oxygenases.

Main Methods:

• A high-throughput aerobic growth selection platform was established in Escherichia coli (strain MX203) to screen for CHMO variants with improved thermostability.
• Random mutagenesis libraries of CHMO were generated and screened at elevated temperatures (42 °C) using cyclohexanone as the substrate.
• Enzyme activity was indirectly measured by the restoration of cellular redox balance through nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes.
• Molecular modeling was employed to understand the structural basis for enhanced thermostability in identified CHMO variants.

Main Results:

• A thermostable CHMO variant, CHMO GV (A245G-A288V), was identified with retained native activity, exhibiting a ~4.4-fold increase in residual activity after incubation at 30 °C.
• CHMO GV demonstrated a ~5-fold higher cyclohexanone conversion rate at 37 °C compared to the wild-type enzyme.
• Molecular modeling revealed improved residue packing and additional backbone hydrogen bonding in CHMO GV, contributing to its enhanced stability.
• Further rational design led to CHMO A245G-A288V-T415C, which showed improved thermostability at 45 °C.

Conclusions:

• The developed high-throughput screening platform enables rapid discovery of thermostability-enhancing mutations for oxygenases like CHMO.
• Engineered CHMO variants exhibit significantly improved thermal stability and activity, addressing a key limitation for industrial biocatalysis.
• The successful protein engineering of CHMO demonstrates the potential for scaling up biocatalytic processes through enhanced enzyme robustness.