Chromosome-level genome assembly of <i>Cornus officinalis</i> reveals the evolution of loganin biosynthesis

<i>Cornus officinalis</i> is a traditional medicinal plant known for producing loganin, a bioactive iridoid glycoside with potential anticancer properties. However, the absence of a high-quality reference genome has limited insights into its biosynthetic pathways. Here, we present a chromosome-level genome assembly of <i>C. officinalis</i> with a size of 2.85 Gb. Comparative genomic analysis revealed that the genome expansion and longer gene structures, relative to other <i>Cornales</i> species, are primarily due to a recent expansion of transposable elements. In this study, we identified unique biosynthetic gene clusters coding multiple core enzymes, including loganin acid O-methyltransferase (LAMT), secologanin synthase (SLS), and cytochrome P450, all of which catalyze sequential steps leading to loganin formation. LAMT enzymes from <i>C. officinalis</i> capable of catalyzing the C-9 hydroxylation of loganin acid were identified, whereas the homolog (CoLMAT) was not found to possess this activity. Additionally, molecular docking studies revealed critical residues in CoLAMT that govern substrate positioning, providing insights into the mechanism of C-9 regioselective hydroxylation. Further characterization of 7-deoxyloganicacid hydroxylase, LAMT, and SLS enzymes allowed us to elucidate the complete biosynthetic pathway of major loganin derivatives in the medicinal plant <i>C. officinalis</i>. Finally, we introduced CoLAMT and its upstream genes into <i>Nicotiana benthamiana</i> and successfully achieved the <i>de novo</i> biosynthesis of a series of loganin derivatives. This work reveals key evolutionary and molecular mechanisms in loganin biosynthesis, providing insights into biotechnological applications in anticancer drug development.