Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

Ping Su,Wei Gao,Meng Zhang,Yuru Tong,Qiqing Cheng,Jian Yang,Luqi Huang,Zhongqiu Teng,Yating Hu

doi:10.1038/srep23057

Abstract

Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes.

Highlights

Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes
The full-length SmCPSent and SmKS cDNAs were determined by 5′ RACE and 3′ RACE, and the corresponding cDNA sequences were submitted to the National Center for Biotechnology Information (Supplementary Fig. S1)
The amino acid sequence contains a conserved DIDD motif (Fig. 3), which strongly suggests that SmCPSent can catalyze GGPP to CPP as a class II diterpene synthases (diTPSs)

Summary

Introduction

Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. We clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes. Salvia miltiorrhiza Bunge has been widely used in China (and to a lesser extent in Japan, the United States, and European countries) for the treatment of cardiovascular and cerebrovascular diseases This medicinal herb exhibits anti-inflammatory, antioxidant and radical scavenging effects[1,2]. Biochemical studies suggested that CPS and KS might interact with one another[10]; we constructed a fused SmCPSent and SmKS protein and showed that the production of ent-kaurene was significantly improved

Methods

Results

Conclusion