The gap-free genome and functional characterization of O-methyltransferases provide insights into the biosynthesis of sanguinarine and chelerythrine in Eomecon chionantha

Benzylisoquinoline alkaloids (BIAs) are a notable class of bioactive natural products with therapeutic potential. Metabolomic profiling identified a total of 186 BIAs across various tissues of Eomecon chionantha, with sanguinarine and chelerythrine being the predominant compounds, quantified at 5.2 mg/g and 9.9 mg/g in the roots, respectively. The biosynthetic pathways for these compounds have been elucidated in species of the Papaveraceae family, where methylation events are crucial. Here, we present a telomere-to-telomere (T2T) gap-free genome assembly of E. chionantha, which has a total size of 368.5 Mb, comprising nine centromeric regions, 15 telomeres, 19,785 protein-coding genes, and 58.89% repetitive sequences. Genome analysis reveals a single whole-genome duplication in E. chionantha predating its divergence from Macleaya cordata (∼37.9 million years ago). Gene family analysis revealed the presence of 28 O-methyltransferase (OMT) genes in the E. chionantha genome, predominantly amplified through tandem duplication events, as well as the screening of EcOMT3/4/5/10/17/25/26/27 may be involved in the biosynthesis of sanguinarine and chelerythrine. Functional characterization demonstrated that all eight EcOMTs exhibit activity as 6OMT and scoulerine-9-O-methyltransferase (SMT), with only EcOMT17 functioning specifically as a 4'OMT, indicating that multiple EcOMTs have the catalytic capacity for 6OMT and SMT functions, while 4'OMT activity is highly specific. Collectively, this work elucidates OMT roles in BIA biosynthesis while offering genomic resources for Papaveraceae research and evolutionary insights into alkaloid diversification.