Integrated strategies for engineering ferulic acid decarboxylase with tunnel conformation and substrate pocket for adapting non-natural substrates

Ferulic acid decarboxylase (Fdc1) catalyzes the decarboxylation of ferulic acid derivatives from lignin with broad substrate spectra. However, the catalytic efficiency of Fdc1 across non-natural substrates with bulky substituents attached to the benzene ring limits its application. Here, an integrated strategy was developed to engineer ScFdc1 from Saccharomyces cerevisiae, specifically targeting 4-acetoxycinnamic acid for the production of 4-acetoxystyrene, which was widely used in photoresists monomers. The strategy entailed the integrating two structural aspects of the enzyme, the conformation of the access tunnel and the substrate binding pocket. Six mutants from a 1248-variant library with significant impacts on enzyme performance were identified. Integration of the two aspects of the mutants achieved the ScFdc1_F397V/I398L/T438P/P441V variant, exhibiting over an 11.8-fold activity improvement towards 4-acetoxycinnamic acid. The variant adopts an open tunnel conformation against that the closed state in the wild type, as revealed by structural analysis with the minimal distance at the bottleneck of the tunnel increased from 0.7 to 1.4 nm. Additionally, a reshaped binding pocket is identified which facilitates the substrate accessibility and binding affinity confirmed by enzymatic and molecular dynamics analysis. The results not only provide strategies for enzyme engineering through the accumulation of beneficial mutants, but also offer a promising route for sustainable styrene derivative production.