Two O -Methyltransferases Mediate Multiple Methylation Steps in the Biosynthesis of Coumarins in Cnidium monnieri .

Coumarins with methoxy groups such as osthole ( 1 ), xanthotoxin ( 2 ), bergapten ( 3 ), and isopimpinellin ( 4 ) are typical bioactive ingredients of many medicinal plants. The methylation steps remain widely unknown. Herein, we report the discovery of two methyltransferases in the biosynthesis of O -methyl coumarins in Cnidium monnieri by transcriptome mining, heterologous expression, and in vitro enzymatic assays. The results reveal that (i) CmOMT1 catalyzes the methylation of osthenol ( 8 ) as the final step in the biosynthesis of 1 , (ii) CmOMT2 shows the highest efficiency and preference for methylating xanthotoxol ( 11 ) to form 2 , and (iii) CmOMT1 and CmOMT2 also efficiently transform bergaptol ( 10 ) and 8-hydroxybergapten ( 13 ) into 3 or 4 , suggesting the CmOMTs mediate multistep methylations in the biosynthesis of linear furanocoumarins in C . monnieri .