Salvia miltiorrhiza (Danshen in Chinese), is a well-known traditional Chinese medicinal plant, which is used as not only human medicine but also health-promotion food. Danshen has been extensively used for the treatment of various cardiovascular and cerebrovascular diseases. As a major group of bioactive constituents from S. miltiorrhiza, water-soluble phenolic acids such as salvianolic acid B possessed good bioactivities including antioxidant, anti-inflammatory, anti-cancer and other health-promoting activities. It is of significance to improve the production of phenolic acids by modern biotechnology approaches to meet the increasing market demand. Significant progresses have been made in understanding the biosynthetic pathway and regulation mechanism of phenolic acids in S.miltiorrhiza, which will facilitate the process of targeted metabolic engineering or synthetic biology. Furthermore, multiple biotechnology methods such as in vitro culture, elicitation, hairy roots, endophytic fungi and bioreactors have been also used to obtain pharmaceutically active phenolic acids from S. miltiorrhiza. In this review, recent advances in bioactivities, biosynthetic pathway and biotechnological production of phenolic acid ingredients were summarized and future prospective was also discussed.

Salvia miltiorrhiza Bunge is an herb rich in bioactive tanshinone and salvianolic acid compounds. It is primarily used as an effective medicine for treating cardiovascular and cerebrovascular diseases. Liposoluble tanshinones and water-soluble phenolic acids are a series of terpenoids and phenolic compounds, respectively. However, the regulation mechanism for the simultaneous promotion of tanshinone and salvianolic acid biosynthesis remains unclear. This study identified a R2R3-MYB subgroup 20 transcription factor (TF), SmMYB98, which was predominantly expressed in S. miltiorrhiza lateral roots. The accumulation of major bioactive metabolites, tanshinones, and salvianolic acids, was improved in SmMYB98 overexpression (OE) hairy root lines, but reduced in SmMYB98 knockout (KO) lines. The qRT-PCR analysis revealed that the transcriptional expression levels of tanshinone and salvianolic acid biosynthesis genes were upregulated by SmMYB98-OE and downregulated by SmMYB98-KO. Dual-Luciferase (Dual-LUC) assays demonstrated that SmMYB98 significantly activated the transcription of SmGGPPS1, SmPAL1, and SmRAS1. These results suggest that SmMYB98-OE can promote tanshinone and salvianolic acid production. The present findings illustrate the exploitation of R2R3-MYB in terpenoid and phenolic biosynthesis, as well as provide a feasible strategy for improving tanshinone and salvianolic acid contents by MYB proteins in S. miltiorrhiza.

Water-soluble phenolic acids are major bioactive compounds in the medicinal plant species Salvia miltiorrhiza. Phenolic acid biosynthesis is induced by methyl jasmonate (MeJA) in this important Chinese herb. Here, we investigated the mechanism underlying this induction by analyzing a transcriptome library of S. miltiorrhiza in response to MeJA. Global transcriptome analysis identified the MeJA-responsive R2R3-MYB transcription factor-encoding gene SmMYB1. Overexpressing SmMYB1 significantly promoted phenolic acid accumulation and upregulated the expression of genes encoding key enzymes in the phenolic acid biosynthesis pathway, including cytochrome P450-dependent monooxygenase (CYP98A14). Dual-luciferase (dual-LUC) assays and/or an electrophoretic mobility shift assays (EMSAs) indicated that SmMYB1 activated the expression of CYP98A14, as well as the expression of genes encoding anthocyanin biosynthesis pathway enzymes, including chalcone isomerase (CHI) and anthocyanidin synthase (ANS). In addition, SmMYB1 was shown to interact with SmMYC2 to additively promote CYP98A14 expression compared to the action of SmMYB1 alone. Taken together, these results demonstrate that SmMYB1 is an activator that improves the accumulation of phenolic acids and anthocyanins in S. miltiorrhiza. These findings lay the foundation for in-depth studies of the molecular mechanism underlying MeJA-mediated phenolic acid biosynthesis and for the metabolic engineering of bioactive ingredients in S. miltiorrhiza.

Phenolic acids and tanshinones are major bioactive ingredients in Salvia miltiorrhiza, which possess pharmacological activities with great market demand. However, transcriptional regulation of phenolic acid and tanshinone biosynthesis remains poorly understood. Here, a basic leucine zipper transcription factor (TF) named SmbZIP1 was screened from the abscisic acid (ABA)-induced transcriptome library. Overexpression of SmbZIP1 positively promoted phenolic acid biosynthesis by enhancing expression of biosynthetic genes such as cinnamate-4-hydroxylase (C4H1). Furthermore, biochemical experiments revealed that SmbZIP1 bound the G-Box-like1 element in the promoter of C4H1 gene. Meanwhile, SmbZIP1 inhibited accumulation of tanshinones mainly by suppressing the expression of biosynthetic genes including geranylgeranyl diphosphate synthase (GGPPS) which was confirmed as a target gene by in vitro and in vivo experiments. By contrast, the phenolic acid content was reduced and tanshinone was enhanced in CRISPR/Cas9-mediated knockout lines. In addition, the previously reported positive regulator SmERF1L1 on tanshinone biosynthesis, was found to be inhibited in overexpression-SmbZIP1 lines indicated by RNA sequencing, and was proven to be the target of SmbZIP1. In summary, this work uncovers a novel regulator and deepens our understanding of the transcriptional and regulatory mechanisms of phenolic acid and tanshinone biosynthesis, and sheds new lights on metabolic engineering in S. miltiorrhiza.