Lineage-Specific Neofunctionalization of Polyacetylene-Directing UGT76 Glycosyltransferases in Campanulaceae

Abstract

Polyacetylene glycosides exhibit notable pharmacological activities, yet the glycosyltransferases acting on their polyacetylene scaffolds remain unknown. Here we report a telomere-to-telomere genome assembly of Codonopsis pilosula and, guided by spatial metabolomics, characterize three UDP-glycosyltransferases: CpUGT76BG1 and CpUGT76BG2 catalyze the direct glycosylation of lobetyol to lobetyolin, while CpUGT94BY2 performs subsequent sugar-sugar coupling to produce lobetyolinin, with each activity confirmed by in planta overexpression. Structural modeling reveals that CpUGT76BG1 and CpUGT76BG2 employ a deep hydrophobic tunnel to fully encase the linear polyacetylene chain, a binding architecture distinct from the shallow pockets used by canonical plant UGTs for planar aromatic substrates. Ancestral sequence reconstruction across eleven nodes partitions the UGT76 lineage into three functionally distinct evolutionary stages, tracing the trajectory from an ancestral shallow pocket to this specialized deep architecture. These findings establish the key glycosylation steps of polyacetylene glycoside biosynthesis, define a tunnel-based paradigm for non-planar substrate recognition, and reveal how tandem duplication-driven active site remodeling generates metabolic novelty.