Structure-Guided Co-Evolution of Fungal Unspecific Peroxygenase for Improved VD₃ C25-Hydroxylation by...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Protein Engineering
• Synthetic Biology

Background:

• Fungal unspecific peroxygenases (UPOs) catalyze selective C-H bond oxyfunctionalization using H2O2.
• Traditional genetic modification and expression in Pichia pastoris are time-consuming, limiting rapid enzyme evolution.

Purpose of the Study:

• To develop a structure-guided combinatorial evolution strategy (SBPCS) to accelerate the engineering of AaeUPO for Vitamin D3 (VD3) C25-hydroxylation.
• To improve the efficiency and yield of 25(OH)VD3 production.

Main Methods:

• Implemented a structure-guided combinatorial evolution strategy (SBPCS) involving multipoint mutagenesis.
• Utilized molecular dynamics (MD) simulations to guide the screening of key residues in the substrate-binding pocket.
• Performed iterative rounds of mutagenesis and screening on AaeUPO.

Main Results:

• Identified a triple mutant (G195A/V244I/S272A, M3) with a 5.16-fold increase in C25-hydroxylation efficiency.
• Achieved a significant increase in 25(OH)VD3 yield from 45.8 to 354.40 mg/L.
• Observed shortened substrate-heme Fe═O distance and stabilized dihedral angle in the M3 mutant.

Conclusions:

• The SBPCS strategy effectively accelerates the coevolution of AaeUPO for enhanced VD3 C25-hydroxylation.
• Enzyme engineering guided by structural insights and MD simulations can optimize biocatalytic efficiency.
• Rational design approaches are crucial for advancing enzyme evolution for specific industrial applications.