Structure-coevolution dual-guided engineering of C-glycosyltransferase enables high-level biosynthesis of vitexin in Yarrowia lipolytica

Flavonoid C-glycosides are bioactive compounds with significant pharmaceutical potential. While biosynthesis offers a sustainable and green alternative to traditional chemical synthesis, its industrial scalability has been hindered by the poor catalytic efficiency and low thermostability of natural C-glycosyltransferases (CGTs). In this study, we report a structure-coevolution dual-guided engineering strategy to modify TcCGT1 from Trollius chinensis, a pivotal enzyme in flavonoid C-glycosylation. The engineered variant M7 exhibited a 43.1-fold improvement in catalytic activity toward apigenin and up to 55.0-fold enhancement for other flavonoid acceptors. It also exhibited an 9.2-fold increase in half-life at 30 °C. Molecular dynamics simulations and structural analyses revealed that the mutations remodeled the substrate-binding cavity and optimized its interactions. To translate this enzymatic advance into a biosynthetic platform, we integrated the optimized variant into a de novo synthetic pathway and conducted metabolic engineering in Yarrowia lipolytica. This integration achieved a vitexin titer of 361.0 mg/L, a 6.3-fold improvement over the wild-type-expressing strain. Furthermore, the engineered microbial cell factory produced 2.14 g/L of vitexin in fed-batch fermentation, demonstrating industrial potential. This study provides new prospects for the structure-coevolution dual-guided synergistic framework of CGT engineering and a sustainable synthetic biology platform for high-value flavonoid C-glycosides.