Structure-Based Pocket Modification to Regulate Substrate Selectivity of a Novel Fatty Acid Photodecarboxylase from Chrysochromulina tobinii

Fatty acid photodecarboxylases (FAPs) are promising biocatalysts for converting renewable fatty acids into alkanes. However, naturally occurring FAPs capable of catalyzing aromatic acids are exceedingly rare. In this study, we engineered the substrate selectivity of a novel FAP from Chrysochromulina tobinii (<i>Ct</i>FAP) by modifying its catalytic pocket structure. The active variant <i>Ct</i>FAP-S endowed it with unique preferential catalysis of 5-phenylvaleric acid (43.1%) over linear C8 and C18 fatty acids (13.5–33.5%). Mutagenesis revealed that sites L431 and F437 are key determinants of the C<i>t</i>FAP-S substrate selectivity. The double mutant L431F/F437W shifted catalytic preference toward long-chain substrates, achieving 77.4% conversion for C18:0. Molecular dynamics confirmed cavity geometry-directed substrate preference modulation. Analogous mutations in other FAPs validated the universal role of catalytic pocket geometry in substrate selectivity regulation. This work provides a structure-guided strategy for tailoring FAP catalytic properties, paving a new avenue for biofuel and fine chemical production.