Flavin-dependent monooxygenases as versatile biocatalysts in biomanufacturing: mechanisms, engineering, and applications.

Flavin-dependent monooxygenases (FMOs) are versatile oxidative biocatalysts that catalyze a wide array of oxygenation reactions, such as hydroxylation, epoxidation, Baeyer-Villiger oxidation, and halogenation. These enzymes utilize flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as cofactors to mediate selective incorporation of oxygen into diverse substrates. Owing to their remarkable chemo-, regio-, and stereoselectivity, FMOs have attracted increasing attention as powerful tools for biomanufacturing. Recent advances in enzyme engineering, structural biology, and computational design have expanded the catalytic diversity of FMOs and enabled their integration into biocatalysis frameworks. Moreover, developments in cofactor regeneration, directed evolution, and cell-free biotransformation have improved FMOs' catalytic efficiency and scalability. Despite these advances, challenges such as limited thermostability, oxygen transfer efficiency, and substrate scope remain obstacles for industrial applications of FMOs. This review summarizes the structural characteristics, catalytic mechanisms, and engineering strategies of FMOs, highlights recent progress in their integration into biocatalysis platforms, and discusses current limitations and possible solutions. Insights into improving FMO catalytic performance and expanding their potential as next-generation biocatalysts for biosynthesis will be provided.