Tanshinones: Leading the way into Lamiaceae labdane-related diterpenoid biosynthesis

Abstract

Tanshinones are the bioactive diterpenoid constituents of the traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), and are examples of the phenolic abietanes widely found within the Lamiaceae plant family. Due to the significant interest in these labdane-related diterpenoid natural products, their biosynthesis has been intensively investigated. In addition to providing the basis for metabolic engineering efforts, this work further yielded pioneering insights into labdane-related diterpenoid biosynthesis in the Lamiaceae more broadly. This includes stereochemical foreshadowing of aromatization, with novel protein domain loss in the relevant diterpene synthase, as well as broader phylogenetic conservation of the relevant enzymes. Beyond such summary of more widespread metabolism, formation of the furan ring that characterizes the tanshinones also has been recently elucidated. Nevertheless, the biocatalysts for the pair of demethylations remain unknown, and the intriguing potential connection of these reactions to the further aromatization observed in the tanshinones are speculated upon here.

Keywords: 2-oxoglutarate dependent dioxygenases; Cytochromes P450; Short-chain alcohol dehydrogenases/reductases; Terpene synthases.

Figure 1.. Tanshinones and their base structures.

Shown are the base structures for labdanes and abietanes, along with the three most prevalent tanshinones found in S. miltiorrhiza (numbered as described in the text)

Figure 2.. The subcellular compartmentalization of isoprenoid and tanshinone biosynthesis more specifically.

AACT, acetoacetyl-CoA thiolase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MDC, pyrophosphate decarboxylase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-Methyl-D-erythritol 4-phosphate; DXR, DXP reductoisomerase; CDP-ME, 4-diphosphocytidyl-2-C-methyl D-erythritol; MCT, 4-diphosphocytidyl-2-C-methyl D-erythritol synthase; CMK, 4-diphosphocytidyl-2-C-methyl D-erythritol kinase; CDP-ME2P, 4-diphosphocytidyl-2-C-methyl D-erythritol 2-phosphate; MEcPP, 2-C-methyl-D-erythritol 2, 4-cyclodiphosphate; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HMBPP, 4-hydroxy-3-methylbut-2-enyl diphosphate; HDS, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IPI, IPP isomerase; GPP, geranyl pyrophosphate; GPPS, geranyl diphosphate synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; GGPP, geranyl geranyl pyrophosphate; GGPPS, geranylgeranyl diphosphate synthase

Figure 3.. Tissue and cell-type specific CPS, KSL and CYP gene expression in S. miltiorrhiza.

Key enzymatic genes highly expressed in root (colored red, consistent with the accumulation of the pigmented tanshinones in this organ), leaf and flower. In addition, the root was further divided into four cross-section cell-types, roughly correlated to the periderm (colored red, consistent with the more specific localization of tanshinones in this cell-type), cortex, phloem and xylem, and those genes known to have higher expression in these cell-types also are indicated. Red text indicates genes with known roles in tanshinone biosynthesis and those known to exhibit inducible expression with an asterisk (*)

Figure 4.. Proposed tanshinone biosynthesis.

Miltiradiene is produced from the general diterpenoid precursor GGPP by the successive action of SmCPS1 and SmKSL1, and undergoes presumably spontaneous oxidation to abietatriene. The ferruginol synthase (CYP76AH1, green), 11-hydroxyferruginol synthase (CYP76AH3, red), and 20-hydroxylase (CYP76AK1, blue) then catalyze successive hydroxylation reactions at C12, C11, C7, and C20, respectively. CYP71D375 (orange) is known to generate the 14,16-epoxy D-ring, which is then further oxidized to a furan ring by Sm2ODD14 (yellow). Compounds numbered as described in the text, while the shaded area indicates the linear pathway that seems to predominate in planta biosynthesis

Figure 5.. Phylogeny, protein and genomic structure of CPS and KSL genes in S. miltiorrhiza.

Left) Phylogenetic tree of diterpene cyclases and synthases from Salvia miltiorrhiza (Sm), Salvia splendens (Ss), Salvia fruticosa (Sf), Rosemarinus officinalis (Ro) and Arabidopsis thaliana (At), constructed using the neighbor-joining method with MEGA-X. Numbers at nodes indicate bootstrap values (percentage of 1000 replicates). The scale bar at bottom represents the genetic distance. Middle) Domain architecture (box coloring: white, plastid targeting sequence; orange, γ domain; green, β domain; blue, α domain). Right) Exon/intron structure of the indicated genes. Blue boxes indicate exons and black lines indicate introns

Figure 6.. Chromosomal location of tanshinone biosynthesis genes in S. miltiorrhiza and comparison of associated BGC with S. splendens.

(a) S. miltiorrhiza chromosomes with position of the tanshinone (ferruginol) associated biosynthetic gene cluster (BGC) as well as that of the CYP71D array and individual genes (CPS and CYP76AK1–3) indicated by a red arrow and black triangles, respectively. (b) Syntenic relationship of the Danshen BGC and CYP71D tandem array with S. splendens. Gene nomenclature follows that previously assigned [9,58,60,66]