Engineering the substrate scope of the thermostable phenolic acid decarboxylase N31 towards sterically hindered phenolic acids

Phenolic acid decarboxylases (PADs) have significant potential for converting bio-based hydroxycinnamic acids (e.g. ferulic acid, p-coumaric acid, caffeic acid, and sinapic acid) into valuable hydroxystyrene monomers. These monomers are in high demand in various industries, including additives in polymer production, cosmetics, and flavoring. PADs offer an efficient and scalable method for producing these compounds under mild reaction conditions. Establishing a viable industrial process requires a thermostable enzyme that shows robustness under operational conditions. Therefore, in this study, we assessed five thermostable ancestors towards their activity and stability for conversion of ferulic acid and sinapic acid at different temperatures. A combinatorial active site library was prepared for the most thermostable ancestor. Among the diverse hydroxystyrene monomers, especially 4-vinyl syringol (4-VS) the decarboxylation product from sinapic acid, exhibits interesting properties. Its polymers demonstrate similar thermal stability characteristics and higher glass transition temperatures compared to those based on vinyl guaiacol, the decarboxylation product from ferulic acid. For this potential, we expanded the substrate scope of the selected PAD ancestor to include sinapic acid through directed mutagenesis. A trade-off between ferulic/caffeic acid and sinapic acid was observed and further investigated via molecular dynamics simulations. The most thermally stable ancestor was identified with a half-life time of 3.65 days, analyzed at 50 °C. We found the Ile29Ser-Leu80Ser-Ile93Ala triple mutation (SSA) to effectively expand the substrate scope with an almost 7-fold increase in activity for sinapic acid, with a half-life time of 1.12 days at 50 °C, being approximately 1,610-fold higher than the PAD from Bacillus subtilis.