Engineering of ω-transaminases for advanced applications.

ω-Transaminases (ω-TAs) are well-established biocatalysts for the asymmetric synthesis of chiral amines, yet their broader application remains limited by challenges related to activity, substrate scope, equilibrium constraints in asymmetric synthesis, and process compatibility e.g. solvent tolerance. In recent years, research has shifted from enzyme discovery toward targeted development strategies aimed at addressing these limitations. This review summarizes recent advances in ω-transaminase development, with a focus on protein engineering and process-level optimization. Structure-guided and semi-rational mutagenesis approaches continue to provide robust, incremental improvements, while multi-site, activity-driven engineering has proven effective for challenging substrates such as β- and γ-amino acids or bulky amines. Emerging AI- and AlphaFold-assisted workflows increasingly support rational enzyme design by reducing experimental efforts. Beyond enzyme sequence optimization, reaction engineering, multienzyme cascades, and process intensification strategies play a crucial role in improving overall performance and scalability. Genome mining and discovery efforts are briefly discussed as supporting tools for expanding the ω-transaminase toolbox. Overall, studies in the last years show that successful ω-transaminase development relies more and more on integrated approaches combining enzyme engineering with intelligent process design, rather than on isolated optimization strategies. KEY POINTS: •The state-of-the-art status of ω-TA development is presented. •The shift from enzyme discovery to substrate-based optimization is highlighted. •Modern reaction conditions for more environmentally friendly synthesis are reported.