Expansion within the CYP71D subfamily drives the heterocyclization of tanshinones synthesis in Salvia miltiorrhiza

Abstract

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza). These groups of chemicals have the characteristic furan D-ring, which differentiates them from the phenolic abietane-type diterpenoids frequently found in the Lamiaceae family. However, how the 14,16-epoxy is formed has not been elucidated. Here, we report an improved genome assembly of Danshen using a highly homozygous genotype. We identify a cytochrome P450 (CYP71D) tandem gene array through gene expansion analysis. We show that CYP71D373 and CYP71D375 catalyze hydroxylation at carbon-16 (C16) and 14,16-ether (hetero)cyclization to form the D-ring, whereas CYP71D411 catalyzes upstream hydroxylation at C20. In addition, we discover a large biosynthetic gene cluster associated with tanshinone production. Collinearity analysis indicates a more specific origin of tanshinones in Salvia genus. It illustrates the evolutionary origin of abietane-type diterpenoids and those with a furan D-ring in Lamiaceae.

Analysis of the Coptis chinensis genome reveals the diversification of protoberberine-type alkaloids

“…We followed the method recently reported in Salvia for our biochemical analysis 95 . Briefly, the epitope-tagged pESC-His vectors carrying CYP719 genes were each transformed into the yeast strain WAT11.…”

The ERF‐VII transcription factor SmERF73 coordinately regulates tanshinone biosynthesis in response to stress elicitors in Salvia miltiorrhiza

“…With the completion of the Danshen draft genome (Xu et al., 2016; Ma et al., 2021), and now that elicitor‐responsive transcriptomic data are available (Gao et al., 2014; Luo et al., 2014; Zhang et al., 2016; Zhou et al., 2017) many transcription factors have been found to be implicated in the regulation of tanshinone biosynthesis. However, analyses have mostly concentrated on the genes encoding enzymes in the terpenoid precursor and the carbon skeleton formation stages.…”

Recent progress and new perspectives for diterpenoid biosynthesis in medicinal plants

“…Gene clusters in the diterpenoid pathway and the coordinated expression of proteins in response to environmental stimuli or elicitation have also been observed 79,80 . In the genome, gene family expansion and gene clusters of novel P450 multigene subfamilies for secondary metabolic pathways have been observed in certain plant orders, families, or species 5,21,22,81 . This limited distribution suggests that phylogenetic analysis of genes in clusters could facilitate the discovery and characterization of plant P450s involved in highly specialized pathways, although it is worth keeping in mind the possibility that genes encoding P450s may not always be clustered with genes encoding the relevant terpene synthases.…”

“…Recently, by gene expansion analysis, a P450 tadem gene array was identified. Two of them (CYP71D373 and CYP71D375) were characterized to catalyze hydroxylation and heterocyclization to form the d ‐ring of tanshinones 81 . Based on genome sequencing and comparative omics data, several promising P450s and 2ODDs have been identified as candidates that complete the final steps in the biosynthetic pathway for these active compounds 80,98 …”

“…However, the synthetic biology‐engineered production of diterpenoids by microorganisms is always challenged by the multiple steps requiring P450‐mediated oxidation and other modifications necessary for the biosynthesis of complex diterpenoids. For example, there are at least eight P450s involved in the biosynthesis of paclitaxel, 88 and at least four P450s are involved in the biosynthesis of tanshinones 69,72,81 . In this case, yeast, with its internal membrane system and native mevalonate pathway, has assumed an essential role in the production of diterpenoids.…”