Computational and deep-learning-guided design of raspberry ketone synthase towards concurrent gains in activity and...

Raspberry ketone synthase is the key enzyme for raspberry ketone biosynthesis, yet its activity and thermostability remain suboptimal for industrial use. By combining computational and deep-learning-guided design, we obtained a Rubus idaeus raspberry ketone synthase (RiRZS1) mutant M3 with 12.06-fold higher relative activity and 38.71-fold improved thermostability compared with the wild type. Kinetic analysis showed that M3 exhibited 14.89-fold higher k_cat and 5.91-fold higher catalytic efficiency(k_cat/K_m), indicating markedly enhanced catalytic competence. Structural inspection and molecular dynamics simulations revealed that M3 induced only subtle conformational rearrangements but reduced global flexibility, accounting for its improved thermostability. Meanwhile, tunnel and substrate-pocket reshaping in M3 was associated with increased prereaction-state populations, which may contribute to enhanced catalytic performance. In whole-cell biotransformation, the raspberry ketone titer of M3 was 2.38-fold that of wild-type, and the corresponding space-time yield was 3.57-fold higher. This computational and artificial intelligence-assisted framework enabled a synergistic optimization of RiRZS1 activity and stability, and can serve as a transferable design framework for protein engineering in sustainable bio-manufacturing.