Functional characterization of O-methyltransferases reveals the stepwise biosynthetic mechanism of erianin in Dendrobium chrysotoxum

Erianin is a bibenzyl compound with remarkable anti-tumor activity, abundantly present in the medicinal orchid Dendrobium chrysotoxum Lindl. However, the precise enzymatic steps governing erianin biosynthesis in D. chrysotoxum remain partially uncharacterized, limiting its potential for medicinal development and utilization. In this study, the accumulation dynamics of erianin were systematically tracked across different developmental stages of D. chrysotoxum stems. An inverse accumulation pattern was observed between erianin and another major bibenzyl, gigantol, during stem development. Metabolomic analysis of stems at three representative developmental stages led to the proposal of a six-step biosynthetic pathway for erianin, in which four steps are catalyzed by O-methyltransferases (OMTs), underscoring their central role. A comparative transcriptomic analysis identified 17 candidate OMT genes, three of which were confirmed to have the expected function through in vitro and in vivo enzyme activity assays and tobacco transient transformation system validation experiments. Furthermore, the three identified DchOMTs exhibit distinct substrate specificities, with each one catalyzing a different step in the erianin biosynthetic pathway. Molecular docking further supported the stable binding affinity between each OMT and its corresponding substrate. This study elucidates key molecular mechanisms involved in erianin biosynthesis and provides a genetic explanation for its high accumulation in D. chrysotoxum . The findings offer valuable targets and gene resources for the metabolic engineering and synthetic biology of bibenzyl-derived anticancer natural products.