Reprogramming a Baeyer-Villiger monooxygenase for improved synthesis of 3-hydroxypropionic acid precursor by...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Green Chemistry

Background:

• Baeyer-Villiger monooxygenases (BVMOs) are crucial biocatalysts for producing 3-hydroxypropionic acid (3-HP) precursors from alkyl levulinates.
• Current BVMO applications are hindered by poor operational stability, side reactions, and limited control over product formation.

Purpose of the Study:

• To engineer the Acinetobacter radioresistens Baeyer-Villiger monooxygenase (Ar-BVMO) for enhanced butyl levulinate oxidation.
• To improve BVMOs for sustainable synthesis of 3-HP precursors via structure-guided hotspot transfer.

Main Methods:

• Structure-guided hotspot transfer strategy inspired by cyclopentanone monooxygenase (CPMO).
• Generation and characterization of Ar-BVMO mutants (Y141L, Y142L) using molecular dynamics simulations, kinetic analysis, and whole-cell biotransformations.

Main Results:

• The Y142L mutant exhibited a 2.5-fold increase in catalytic efficiency and a 54% increase in total turnover number.
• The Y141L mutant demonstrated complete regioselectivity but lower productivity.
• Y142L achieved >95% substrate consumption within 24 hours in whole-cell systems.

Conclusions:

• Hotspot transfer to the Ar-BVMO Y141/Y142 region successfully enhanced butyl levulinate oxidation.
• Engineering efforts can decouple enzyme productivity from regioselectivity, enabling targeted optimization for 3-HP precursor synthesis.