Genome evolution and glycosyltransferase diversification shape asperosaponin Ⅵ biosynthesis in Dipsacus asperoides

Deciphering the genomic and enzymatic features associated with high-abundance specialized metabolism is important for understanding and engineering bioactive natural product biosynthesis. Asperosaponin Ⅵ, a hederagenin-based saponin, accumulates at exceptionally high levels in Dipsacus asperoides roots and is by far the predominant triterpenoid saponin. Here, by integrating a chromosome-level genome assembly with comparative genomics, transcriptomics, metabolite profiling, and biochemical characterization, we reconstructed a major biosynthetic route to asperosaponin Ⅵ and identified diversification of triterpenoid-associated gene families. Functional analyses revealed an ordered glycosylation sequence in which DaUGT73GZ2 preferentially accepts C-3-glycosylated substrates retaining a free C-28 carboxyl group, thereby establishing a C-3-before-C-28 glycosylation order during asperosaponin Ⅵ biosynthesis. Phylogenetic and biochemical analyses further uncovered functional differentiation among UGT85CA enzymes involved in C-3 arabinosylation, while genomic and expression analyses revealed lineage-specific patterns of pathway gene duplication and transcriptional regulation. Together, these findings identify genomic, enzymatic, and transcriptional features associated with the distinctive, high-abundance triterpenoid saponin profile of D. asperoides roots and provide a framework for understanding and engineering triterpenoid saponin biosynthesis.