Integrated genomic, transcriptomic, and metabolomic analyses of <i>Ilex hylonoma</i> provide insights into the triterpenoid saponin biosynthesis

SUMMARY Ilex is known for its rich content of secondary metabolites, particularly triterpenoid saponins. These compounds hold significant value in natural remedies and herbal medicine. However, the molecular mechanisms responsible for triterpenoid biosynthesis in plants of this genus remain largely unexplored. In this study, we successfully generated the first chromosome‐scale genome of Ilex hylonoma . The assembly, comprising 20 anchored chromosomes, has an N50 contig size of 2.13 Mb and a scaffold size of 33.68 Mb. Comparative genome analyses with two other congeners with available chromosome‐level genomes suggested that an end‐to‐end chromosome fusion event likely contributed to the reduction in chromosome number from n = 20 to n = 19 within this genus. By integrating transcriptomic and metabolomic data, we identified the gene expression patterns and metabolite profiles of I. hylonoma across three commonly utilized medicinal tissues. We subsequently pinpointed candidate genes involved in the regulation of triterpenoid saponin biosynthesis, including CYP450 genes, UGT genes, and associated transcription factors. Furthermore, yeast heterologous expression analysis revealed that ihyl08363 catalyzed the conversion of β‐amyrin into oleanolic acid, while ihyl04303 catalyzed the C‐2α hydroxylation of oleanolic acid to produce maslinic acid. This integrated analysis provides valuable insights into the biosynthesis of important triterpenoid saponins in medicinal Ilex plants.