Engineering a Rieske-Type Oxygenase Enables O -Demethylation of Ferulic Acid to Synthesize Caffeic Acid

Abstract Ferulic acid is a crucial lignin-derived component that serves as a feedstock for the synthesis of multiple high-value compounds through biological conversion. However, the absence of a ferulate O-demethylase restricts wider applications of ferulate. In this study, a Rieske oxygenase, vanillate O-demtheylase (VanAB), which catalyzes vanillate O-demethylation, was used to achieve ferulate O-demethylation via enzyme engineering. Based on the structure of wild-type VanA, the key amino acid residues at the catalytic site were substituted with residues bearing small R groups to allow ferulate to enter. After screening multiple mutants, the VanAQ167A/D171A mutant exhibited the best catalytic performance toward ferulate. Structural insights revealed that the mutant VanAQ167A/D171A had a broadened catalytic site that allowed ferulate entry. Future engineering directions were proposed based on further structural analyses. This study provides a potential route for the biological valorization of ferulic acid to produce caffeic acid and offers valuable insights for engineering Rieske-type oxygenases.