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

Phenolic acid decarboxylases (PADs) convert bio-based hydroxycinnamic acids into valuable hydroxystyrene monomers under mild reaction conditions. These compounds are in high demand in polymer production, cosmetics, and flavoring. Especially 4-vinyl syringol, the decarboxylation product from sinapic acid, generates polymers with similar thermal stability and higher glass transition temperatures than vinyl guaiacol, the decarboxylation product from ferulic acid. However, natural PAD enzymes typically show slow turnover with sinapic acid. In addition, establishing a viable industrial process requires enzymes operating under elevated temperatures. To tackle these issues, we assessed five thermostable ancestral PADs towards their activity and stability for the conversion of ferulic acid and sinapic acid at different temperatures. A combinatorial active site library was prepared for the most thermostable ancestor. We expanded the substrate scope of a selected PAD ancestor to include sinapic acid through directed mutagenesis. A trade-off between ferulic-/caffeic acid and sinapic acid was observed and investigated via molecular dynamics simulations. The most stable ancestor was identified with a half-life 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 11-fold increase in catalytic efficiency for sinapic acid, and a half-life of 1.12 days at 50°C, being approximately 1.6-fold higher than the frequently used PAD from Bacillus subtilis.