The 2-oxoglutarate-dependent dioxygenase superfamily participates in tanshinone production in Salvia miltiorrhiza

Highly oxidized tanshinones are pharmacological ingredients extracted from the medicinal model plant Salvia miltiorrhiza and are mainly used to treat cardiovascular diseases. Previous studies have confirmed that cytochrome P450 mono-oxygenases (CYP450s) have a key function in the biosynthesis of tanshinones; however, no solid evidence links oxidation to the 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily. Here, we identified 132 members of the DOXB and DOXC subfamilies of 2OGD by scanning the 2OG-FeII Oxy domain using a genome-wide strategy in S. miltiorrhiza. The DOXC class was phylogenetically divided into twelve clades. Combining phylogenetic relationships, differential expression and co-expression from various organs and tissues revealed that two 2OGDs were directly related to flavonoid metabolism, and that 13 2OGDs from different clades were predicted to be involved in tanshinone biosynthesis. Based on this insight into tanshinone production, we experimentally detected significant decreases in miltirone, cryptotanshinone, and tanshinone IIA (0.16-, 0.56-, and 0.56-fold, respectively) in 2OGD5 RNAi transgenic lines relative to the control lines using a metabonomics analysis. 2OGD5 was found to play a crucial role in the downstream biosynthesis of tanshinones following the hydroxylation of CYPs. Our results highlight the evolution and diversification of 2OGD superfamily members and suggest that they contribute to the complexity of tanshinone metabolites.