Polymethoxyflavones (PMFs) are a distinct class of plant flavonoids with significant therapeutic potential, particularly as anticancer agents. This potent bioactivity is largely attributable to O-methylation. However, a major bottleneck for the industrial-scale production of PMFs is the lack of suitable precursors and available O-methyltransferases (OMTs), which are essential for constructing a viable biosynthetic pathway. Here, we discovered a caffeic acid O-methyltransferase (CsOMT5) whose transcriptional dynamics closely mirrored the PMF levels in the flavedo of sweet orange (Citrus sinensis). In vivo validation further verified that CsOMT5 indeed promoted PMF accumulation in citrus. And its recombinant protein possessed broad substrate promiscuity for various PMF intermediates. Notably, CsOMT5 exhibited specialized C5-regioselectivity for naringenin, a readily available precursor for de novo synthesis of PMFs. Furthermore, critical residues involved in this methylation activity (N14, T18, I120, I256, and G305) were revealed through mutational analysis. This study not only provides deeper insights into the PMF biosynthetic pathway but also opens new avenues for the industrial biosynthesis of PMFs.

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