Chromosome‐Level Genome Assembly of the Allotetraploid <i>Gynostemma pentaphyllum</i> Provides Novel Insights Into the Biosynthesis of Ginsenoside and Gypenoside <scp>LVI</scp>

Gynostemma pentaphyllum, a herb used in tea and traditional Chinese medicine, shows geographic variation in its production of valuable dammarane-type ginsenosides and gypenoside LVI between populations from Suining (SN) and Nanning (NN). To elucidate the mechanisms underlying this differential metabolite accumulation, a chromosome-level genome for G. pentaphyllum (SN population) was assembled. The analysis revealed that SN is a tetraploid (~1.2 Gb), resulting from a recent whole-genome duplication event in a diploid ancestor. Phylogenetic analysis indicates SN and diploid NN share a recent common ancestor, diverging approximately 4.95 million years ago. Chromosome evolution analysis confirmed SN is an allotetraploid with clear subgenomic differentiation. This genome, combined with multi-omics data, enabled the screening of candidate P450 genes involved in ginsenoside/gypenoside LVI biosynthesis. In vivo and in vitro experiments confirmed that GpCYP88AB3 functions as a bifunctional enzyme by first hydroxylating dammarenediol-II at C-12 to yield protopanaxadiol (PPD), and then hydroxylating PPD at C-2 to form 2α-OH-PPD. Phylogenetically, GpCYP88AB3 and similar enzymes from Araliaceae belong to distinct CYP subfamilies, demonstrating convergent evolution of this function between the two plant families and highlighting the functional plasticity of P450s. Evolutionary analysis suggests that GpCYP88AB3 emerged from a CYP88 gene family expansion in the tetraploid G. pentaphyllum. This expansion occurred after, but was not directly caused by, the whole-genome duplication event. This study elucidates the biosynthetic pathway for the key metabolites in G. pentaphyllum, providing a foundation for future metabolic engineering and synthetic biology applications.