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

Ying Ma,Luqi Huang,Juan Guo,Xiaoquan Qi,Wen Zeng,Tong Chen,Xiaohui Ma,Yanan Wang,Guanghong Cui,Jian Yang,Yujun Zhao,Changjiangsheng Lai,Baolong Jin,Ruishan Wang,Reuben J Peters,Ye Shen,Jinfu Tang,Liping Kang

doi:10.1038/s41467-021-20959-1

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.

Highlights

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza)
The results reported above further increase the number of genes associated with tanshinone biosynthesis, with three diterpene synthases, SmCPS1, SmCPS2, and SmKSL112,22, and six CYPs, CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, CYP71D375, and CYP71D41111,13
While tanshinone biosynthesis has been investigated for many years[10,11,12,13], the origin of this heterocycle has remained unknown

Summary

Introduction

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. 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. Tanshinones are nor-diterpenoids that form the lipophilic bioactive constituents of Danshen (Fig. 1a)[2,3] These are phenolic abietane-type diterpenoids, which are widely found in the Lamiaceae family[4]. Copalyl diphosphate synthase SmCPS1, with subsequent further cyclization and rearrangement catalyzed by the class I diterpene synthase SmKSL1, which produces the abietane miltiradiene[12]

Methods

Results

Conclusion