Enabling microbial syringol conversion through structure-guided protein engineering

Area of Science:

• Biochemistry
• Microbiology
• Biotechnology

Background:

• Lignin valorization via microbial conversion of aromatic compounds is a promising strategy.
• O-aryl-demethylation is a critical step in aromatic catabolism, converting methoxy groups in lignin to diols for further processing.
• The cytochrome P450 system GcoAB can demethylate guaiacol but not syringol, limiting the microbial breakdown of sinapyl alcohol-derived lignin.

Purpose of the Study:

• To engineer the GcoAB system for efficient syringol O-demethylation.
• To enable microbial utilization of syringol, a component derived from sinapyl alcohol-based lignin.
• To investigate the structural basis for syringol binding and demethylation by engineered GcoAB.

Main Methods:

• Structure-guided protein engineering of the GcoAB system.
• Site-directed mutagenesis of a phenylalanine residue (GcoA-F169) in the GcoAB active site.
• Crystallography and molecular dynamics simulations to analyze protein-ligand interactions.
• In vivo syringol turnover assays in *Pseudomonas putida* KT2440.

Main Results:

• Mutation of GcoA-F169 to smaller amino acids, such as alanine (GcoA-F169A), resulted in efficient syringol O-demethylation.
• Crystallography revealed a productive binding pose of syringol in the engineered variant.
• Molecular dynamics simulations indicated that the mutation eliminated steric clashes, facilitating syringol binding.
• The engineered GcoA-F169A variant demonstrated in vivo syringol turnover in *Pseudomonas putida*.

Conclusions:

• Protein engineering can enhance the substrate specificity of cytochrome P450 enzymes for lignin valorization.
• The engineered GcoAB system enables microbial catabolism of syringol, opening pathways for sinapyl alcohol-derived lignin utilization.
• Cytochrome P450 aromatic O-demethylases show significant potential and plasticity for biological conversion of lignin-derived compounds.