Genome-wide identification of O-methyltransferase genes in Stephania yunnanensis and their roles in benzylisoquinoline alkaloid biosynthesis

Benzylisoquinoline alkaloids (BIAs) are structurally diverse natural products known for their significant pharmacological activities. Although O-methyltransferases (OMTs) play a crucial role in diversifying BIA structures, little is known about the functions of most OMTs in Stephania yunnanensis. This study reports the first genome-wide characterization of the OMT gene family in S. yunnanensis, leading to the identification of 72 SyOMT members in this BIA-producting plant. Seven SyOMT genes located in a cluster on chromosome 11 were cloned and functionally characterized. Enzymatic assays revealed that SyOMT50 and SyOMT52 display broad substrate promiscuity and regioselectivity, catalyzing O-methylation at C6, C7, and C2 positions across various BIA scaffolds, including 1-benzylisoquinolines and tetrahydroprotoberberines. Additionally, SyOMT59 exhibited versatile catalytic activity toward various BIA substrates at multiple sites. Subcellular localization studies indicated that three SyOMTs are localized to the plasma membrane and endoplasmic reticulum. This study reveals the functional roles of SyOMTs in S. yunnanensis and their significance in BIA diversification, providing novel genetic resources for the sustainable synthesis of high-value BIAs.