Unraveling the hydroxylation and methylation mechanism in polymethoxylated flavones biosynthesis in Dracocephalum moldavica.

Dracocephalum moldavica, which belongs to the Lamiaceae family, is an important medicinal plant rich in polymethoxylated flavones (PMFs). PMFs have multi-methoxy groups on the central flavone skeleton. Hydroxylation and methylation are important modifications in the biosynthesis of PMFs, but the corresponding mechanism and related enzymes have not yet been elucidated in D. moldavica. In this study, we analyzed the transcriptome database of D. moldavica and identified three flavonoid hydroxylases (DmFH1/2/3) and four flavonoid O-methyltransferases (DmOMT1/2/3/4) related to the biosynthesis of PMFs. DmFH1 was characterized as a novel F6/8/3'H and has broad substrate specificity for flavones and flavanones. It catalyzes the formation of 7-methylscutellarein and isoscutellarein, which are the key intermediates in PMF biosynthesis. DmOMT4 and DmOMT1 sequentially catalyze the two-step methylation of scutellarein to generate circimaritin, while DmOMT3 has high specificity for 4'-OH of flavonoids. Notably, it catalyzes the conversion of circimaritin into salvigenin, which is an important polymethoxylated flavone. In addition, through heterologous expression of DmFH1 and DmOMT1 in Nicotiana benthamiana, diversified polyhydroxylated and polymethylated metabolites, including 7-methylscutellarein and circimaritin were achieved. Our work uncovers the key hydroxylation and the complex metabolic network of methylation processes in the biosynthesis of PMFs in D. moldavica, and the screened candidate genes can be exploited in synthetic biology research on PMFs.