Structural insights revealed by crystal structures of CYP76AH1 and CYP76AH1 in complex with its natural substrate.

CYP76AH1 is the key enzyme in the biosynthesis pathway of tanshinones in Salvia miltiorrhiza, which are famous natural products with activities against various heart diseases and others. CYP76AH1 is a membrane-associated typical plant class II cytochrome P450 enzyme and its catalytic mechanism has not to be clearly elucidated. Structural determination of eukaryotic P450 enzymes is extremely challenging. Recently, we solved the crystal structures of CYP76AH1 and CYP76AH1 in complex with its natural substrate miltiradiene. The structure of CYP76AH1 complexed with miltiradiene is the first plant cytochrome P450 structure in complex with natural substrate. The studies revealed a unique array pattern of amino acid residues, which may play an important role in orienting and stabilizing the substrate for catalysis. This work would provide structural insights into CYP76AH1 and related P450s and the basis to efficiently improve tanshinone production by synthetic biology techniques.

Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism.

BACKGROUND: The contribution of mitogen-activated protein kinase (MAPK) cascades to plant growth and development has been widely studied, but this knowledge has not yet been extended to the medicinal plant Salvia miltiorrhiza, which produces a number of pharmacologically active secondary metabolites. RESULTS: In this study, we performed a genome-wide survey and identified six MAPKKK kinases (MAPKKKKs), 83 MAPKK kinases (MAPKKKs), nine MAPK kinases (MAPKKs) and 18 MAPKs in the S. miltiorrhiza genome. Within each class of genes, a small number of subfamilies were recognized. A transcriptional analysis revealed differences in the genes' behaviour with respect to both their site of transcription and their inducibility by elicitors and phytohormones. Two genes were identified as strong candidates for playing roles in phytohormone signalling. A gene-to-metabolite network was constructed based on correlation analysis, highlighting the likely involvement of two of the cascades in the synthesis of two key groups of pharmacologically active secondary metabolites: phenolic acids and tanshinones. CONCLUSION: The data provide insight into the functional diversification and conservation of MAPK cascades in S. miltiorrhiza.

Proteomic analysis reveals novel insights into tanshinones biosynthesis in Salvia miltiorrhiza hairy roots.

Salvia miltiorrhiza is a medicinal plant highly appreciated by its content of tanshinones and salvianolic acids. Tanshinones are of particular relevance for their anti-oxidant, anti-tumoral and anti-inflammatory properties. Abiotic and biotic agents as silver nitrate and yeast extract have shown efficiently to stimulate tanshinone accumulation, but the underlying molecular mechanism remains essentially unknown. By using hairy roots as experimental material and the elicitors mentioned, were obtained up to 22 mg of tanshinones per gram of dry weight. Differential label-free quantitative proteomic analysis was applied to study the proteins involved in tanshinone biosynthesis. A total of 2650 proteins were identified in roots extracts, of which 893 showed statistically (p < 0.05) significant change in relative abundance compared to control roots, 251 proteins were upregulated and 642 downregulated. Among the upregulated proteins the predominant functional categories were metabolism (47%), stress defense (18%) and redox homeostasis (10%). Within the metabolism category, isoprenoid metabolism enzymes, cytochromes P450 and FAD-binding berberine proteins showed abundance profile linked to tanshinone concentration. The results presented here allowed to propose 5 new cytochromes P450 and 5 berberine enzymes as candidates to be involved into tanshinone biosynthesis, a novel finding that opens new avenues to improve tanshinone production through biotechnological approaches.

Enhanced production of tanshinone IIA in endophytic fungi Emericella foeniculicola by genome shuffling.

CONTEXT: Tanshinone IIA, commercially produced from Salvia miltiorrhiza Bunge (C.Y.Wu) (Labiatae), has various biological benefits. Currently, this compound is mainly extracted from plants. However, because of the long growth cycle and the unstable quality of plants, the market demands can barely be satisfied. OBJECTIVE: The genomic shuffling technology is applied to screen the high-yield tanshinone IIA strain, which could be used to replace the plant S. miltiorrhiza for the production of tanshinone IIA. The change in the production of tanshinone IIA is clarified by comparing it with the original strain. MATERIALS AND METHODS: Tanshinone IIA was extracted from Strains cells, which was prepared through 0.5 mL protoplast samples by using hypertonic solution I from two different strains. Then, it was analyzed by high-performance liquid chromatography at 30 °C and UV 270 nm. Total DNA from the strains was extracted for RAPD amplification and electrophoresis to isolate the product. RESULTS: In this study, a high-yield tanshinone IIA strain F-3.4 was screened and the yield of tanshinone IIA was increased by 387.56 ± 0.02 mg/g, 11.07 times higher than that of the original strain TR21. DISCUSSION: This study shows that the genetic basis of high-yield strains is achieved through genome shuffling, which proves that genome shuffling can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining the strains with good traits and it is a useful method for the molecular breeding of industrial strains.

Comprehensive transcriptome profiling of Salvia miltiorrhiza for discovery of genes associated with the biosynthesis of tanshinones and phenolic acids.

Tanshinones and phenolic acids are crucial bioactive compounds biosynthesized in Salvia miltiorrhiza. Methyl jasmonate (MeJA) is an effective elicitor to enhance the production of phenolic acids and tanshinones simultaneously, while yeast extract (YE) is used as a biotic elicitor that only induce tanshinones accumulation. However, little was known about the different molecular mechanism. To identify the downstream and regulatory genes involved in tanshinone and phenolic acid biosynthesis, we conducted comprehensive transcriptome profiling of S. miltiorrhiza hairy roots treated with either MeJA or YE. Total 55588 unigenes were assembled from about 1.72 billion clean reads, of which 42458 unigenes (76.4%) were successfully annotated. The expression patterns of 19 selected genes in the significantly upregulated unigenes were verified by quantitative real-time PCR. The candidate downstream genes and other cytochrome P450s involved in the late steps of tanshinone and phenolic acid biosynthesis pathways were screened from the RNA-seq dataset based on co-expression pattern analysis with specific biosynthetic genes. Additionally, 375 transcription factors were identified to exhibit a significant up-regulated expression pattern in response to induction. This study can provide us a valuable gene resource for elucidating the molecular mechanism of tanshinones and phenolic acids biosynthesis in hairy roots of S. miltiorrhiza.

Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast.

The development of medical applications exploiting the broad bioactivities of the diterpene therapeutic triptolide from Tripterygium wilfordii is limited by low extraction yields from the native plant. Furthermore, the extraordinarily high structural complexity prevents an economically attractive enantioselective total synthesis. An alternative production route of triptolide through engineered Saccharomyces cerevisiae (yeast) could provide a sustainable source of triptolide. A potential intermediate in the unknown biosynthetic route to triptolide is the diterpene dehydroabietic acid. Here, we report a biosynthetic route to dehydroabietic acid by transient expression of enzymes from T. wilfordii and Sitka spruce (Picea sitchensis) in Nicotiana benthamiana. The combination of diterpene synthases TwTPS9, TwTPS27, and cytochromes P450 PsCYP720B4 yielded dehydroabietic acid and a novel analog, tentatively identified as 'miltiradienic acid'. This biosynthetic pathway was reassembled in a yeast strain engineered for increased yields of the pathway intermediates, the diterpene olefins miltiradiene and dehydroabietadiene. Introduction in that strain of PsCYP720B4 in combination with two alternative NADPH-dependent cytochrome P450 reductases resulted in scalable in vivo production of dehydroabietic acid and its analog from glucose. Approaching future elucidation of the remaining biosynthetic steps to triptolide, our findings may provide an independent platform for testing of additional recombinant candidate genes, and ultimately pave the way to biotechnological production of the high value diterpenoid therapeutic.

Genetic and correlation analysis of oleoresin chemical components in slash pine.

This is the first comprehensive study of the genetic analysis of the majority of oleoresin components of slash pine (Pinus elliottii). Pine oleoresin, the resin secreted from the pine tree, is a raw material widely used in industrial products. The objective of this study was to explore the genetic variation and correlation between the major oleoresin components of 50 open pollinated families of slash pine. The individual narrow-sense heritability of the 23 oleoresin components and genetic correlations between them were estimated using the residual maximum likelihood in the flexible mixed modeling program, ASReml-R. A high heritability of 0.424 was observed for ß-pinene. Moderate levels of heritability were estimated for ß-phellandrene, methyl abietate, estragole, 15-hydroxy-dehydroabietic acid, and isopimaric acid methyl ester at 0.303, 0.294, 0.27, 0.258, and 0.2, respectively. The heritabilities for pimaric acid methyl ester, abieta-8, 13-diene-18-oic acid methyl ester, sandaracopimaric acid, methyl ester, and camphene were relatively low and ranged from 0.11 to 0.17. Many negative genetic correlations were observed as unfavorable while the corresponding phenotypic correlations presented no significant relationships or positive phenotypic correlations. However, the heritabilities and genetic correlations showed that single or multiple component selections and improvement, directly or indirectly, were effective. We postulate that genetic parameters estimated in this study will work as a reference in breeding programs of oleoresin components, especially in slash pine.

RNA interference targeting CYP76AH1 in hairy roots of Salvia miltiorrhiza reveals its key role in the biosynthetic pathway of tanshinones.

Plant cytochrome P450s (CYPs) are well known as the largest family of enzymes that contribute to both primary metabolism and the chemical diversity of plant secondary metabolites. It is important to elucidate the in vivo role of CYPs in secondary metabolism, in order to apply them in the production of valuable metabolites in medicinal plants via metabolic engineering. CYP76AH1 has been suggested to catalyze the conversion of the carbon skeleton miltiradiene into the intermediate ferruginol, which is involved in the biosynthesis of tanshinones, the chief bioactive ingredients of Salvia miltiorrhiza. However, its role in planta remains to be elucidated. In this work, we constructed a CYP76AH1 RNAi system for hairy roots. Metabolic analysis of RNAi-AH1 hairy root lines showed a significantly increased accumulation of miltiradiene compared to the control lines. At the same time, the concentration of ferruginol decreased revealing the in vivo catalytic activity of CYP76AH1. The content of tanshinones decreased significantly after silencing of CYP76AH1, which verified its key role in the biosynthesis of tanshinones, and indicated that it could be used as a target for metabolic engineering.

Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins.

Jasmonic acid (JA) is an important plant hormone involved in regulation of many aspects of plant growth and development including secondary metabolism and JASMONATE ZIM-DOMAIN (JAZ) proteins are key components in JA signal processes. In this study, two new JAZ genes named SmJAZ3 and SmJAZ9 were cloned from S. miltiorrhiza hairy roots and characterized. Expression profiles under methyl jasmonate (MJ) treatment revealed that SmJAZ3 and SmJAZ9 were both MJ-responsive. Subcellular localization assay showed that SmJAZ3 was located in nucleus while SmJAZ9 was preferentially in nucleus. Expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots differently affected the production of tanshinone. Over-expression of SmJAZ3 or SmJAZ9 in hairy roots produced lower level of tanshinone compared with the control, tanshinone production was as low as 0.077 mg/g DW in line SmJAZ3-3 and 0.266 mg/g DW in line SmJAZ9-22. Whereas, down-regulation of SmJAZs enhanced tanshione production, the content of tanshinone increased to 2.48 fold in anti-SmJAZ3-3 line, and 1.35-fold in anti-SmJAZ9-23 line. Our work indicated that SmJAZ3 and SmJAZ9 are involved in regulation of tanshinone biosynthesis and act as repressive transcriptional regulators in the JA signaling pathway, which paves the way to further dissect molecular mechanism in details in the future.

[Application of synthetic biology to sustainable utilization of Chinese materia medica resources].

Bioactive natural products are the material bases of Chinese materia medica resources. With successful applications of synthetic biology strategies to the researches and productions of taxol, artemisinin and tanshinone, etc, the potential ability of synthetic biology in the sustainable utilization of Chinese materia medica resources has been attracted by many researchers. This paper reviews the development of synthetic biology, the opportunities of sustainable utilization of Chinese materia medica resources, and the progress of synthetic biology applied to the researches of bioactive natural products. Furthermore, this paper also analyzes how to apply synthetic biology to sustainable utilization of Chinese materia medica resources and what the crucial factors are. Production of bioactive natural products with synthetic biology strategies will become a significant approach for the sustainable utilization of Chinese materia medica resources.

[Salvia miltiorrhiza as medicinal model plant].

Research on medicinal model organism is one of the core technologies to promote the modernization of traditional Chinese medicine (TCM). The research progress of Salvia miltiorrhiza as medicinal model plant is summarized in this paper. The genome of S. miltiorrhiza is small and its life cycle is short, as well as this plant can be stably genetically transformed. Because S. miltiorrhiza possesses the important medicinal and economic values, recently the transcriptome and genome of S. miltiorrhiza have been significantly recovered. The research prospect of S. miltiorrhiza as medicinal model plant in TCM was discussed, including biosynthesis of active components and their genetic regulation, relationship between quality of TCM and ecological environments, and selective breeding of good quality lines. Furthermore, as medicinal model plant, the construction of mutant library for S. miltiorrhiza, the genome map with high quality, and the functional genome should be investigated. Accompanying modern investigation of life sciences, the platform for medicinal model plant, S. miltiorrhiza, will be promoted to be established. It is important to develop the ethnopharmacology and new drugs around the world.

Cloning, molecular characterization and functional analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza Bge. f. alba.

The enzyme 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) is a terminal-acting enzyme in the plastid MEP pathway, which produce isoprenoid precursors. The full-length cDNA of HDR, designated SmHDR1 (Genbank Accession No. JX516088), was isolated for the first time from Salvia miltiorrhiza Bge. f. alba. SmHDR1 contains a 1389-bp open reading frame encoding 463 amino acids. The deduced SmHDR1 protein, which shows high identity to HDRs of other plant species, is predicted to possess a chloroplast transit peptide at the N-terminus and four conserved cysteine residues. Transcription pattern analysis revealed that SmHDR1 has high levels of transcription in leaves and low levels of transcription in roots and stems. The expression of SmHDR1 was induced by 0.1 mM methyl-jasmonate (MeJA) and salicylic acid (SA), but not by 0.1 mM abscisic acid (ABA), in the hairy roots of S. miltiorrhiza Bge. f. alba. Complementation of SmHDR1 in the Escherichia coli HDR mutant MG1655 ara < > ispH demonstrated the function of this enzyme. A functional color assay in E. coli showed that SmHDR1 accelerates the biosynthesis of ß-carotene, indicating that SmHDR1 encodes a functional protein. Overexpression of SmHDR1 enhanced the production of tanshinones in cultured hairy roots of S. miltiorrhiza Bge. f. alba. These results indicate that SmHDR1 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza Bge. f. alba.

Overexpression of allene oxide cyclase promoted tanshinone/phenolic acid production in Salvia miltiorrhiza.

KEY MESSAGE: This study provides a desirable candidate gene resource (SmAOC) to increase the content of valuable natural products via appropriate JA pathway genetic engineering. Jasmonates (JAs) are important signal molecules in plants. They regulate transcripts of defense and secondary biosynthetic metabolite genes in response to environmental stresses. Currently, JAs are widely used as elicitors to improve the content of useful secondary metabolism in plants. Synthesis of the naturally occurring enantiomer of various jasmonates is catalyzed by allene oxide cyclase (AOC, EC 5.3.99.6). Here, we cloned and characterized the AOC gene (SmAOC) from Salvia miltiorrhiza. As expected, SmAOC expression was induced by abiotic stimuli such as methyl jasmonate (MeJA), ultraviolet radiation (UV) and low temperature (4 °C) in S. miltiorrhiza plantlets. To demonstrate whether the engineered internal JAs pool by overexpressing AOC gene could promote secondary metabolism production, the SmAOC was incorporated into S. miltiorrhiza hairy roots. The results revealed that SmAOC overexpression significant enhanced the yields of tanshinone IIA, rosmarinic acid (RA) and lithospermic acid B (LAB) in S. miltiorrhiza hairy roots. In addition, expression levels for key genes involved in the biosynthetic pathway of diterpenes and phenolic acids were also altered. These suggest that genetic manipulation of AOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis of JAs.

[Transcriptome characterization for Salvia miltiorrhiza using 454 GS FLX].

To investigate the profile of gene expression in Salvia miltiorrhiza and elucidate its functional gene, 454 GS FLX platform and Titanium regent were used to produce a substantial expressed sequence tags (ESTs) dataset from the root of S. miltiorrhiza. A total of 46 722 ESTs with an average read length of 414 bp were generated. 454 ESTs were combined with the S. miltiorrhiza ESTs from GenBank. These ESTs were assembled into 18 235 unigenes. Of these unigenes, 454 sequencing identified 13 980 novel unigenes. 73% of these unigenes (13 308) were annotated using BLAST searches (E-value < or = 1e-5) against the SwissProt, KEGG TAIR, Nr and Nt databases. Twenty-seven unigenes (encoding 15 enzymes) were found to be involved in tanshinones biosynthesis, and 29 unigenes (encoding 11 enzymes) involved in phenolic acids biosynthesis. Seventy putative genes were found to encode cytochromes P450 and 577 putative transcription factor genes. Data presented in this study will constitute an important resource for the scientific community that is interested in the molecular genetics and functional genomics of S. miltiorrhiza.

The role of a HSV thymidine kinase stimulating substance, scopadulciol, in improving the efficacy of cancer gene therapy.

BACKGROUND: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system. METHODS: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. RESULTS: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC. CONCLUSIONS: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.