Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

Changes in gene expression during hairy root formation by<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> infection in ginseng

Researchers have widely adopted the hairy root culture system as a means for producing secondary metabolites, including ginsenosides from ginseng. Although bacterial genes are involved, the aspects of plant gene expression are unclear. Using a cDNA microarray approach, we identified genes that are differentially expressed in ginseng hairy roots afterAgrobacterium rhizogenes infection. Our goal was to gain an initial understanding of the correlation between hairy root morphology and ginsenoside production. Among the 250 genes analyzed here, 63 (including 14 that are unclassified) were differentially expressed in a hairy root line containing a high level of ginsenosides. Of the genes that had been functionally categorized, 29% and 17% were active in metabolism and stress responses, respectively. Most were primarily associated with ribosomal proteins, thereby functioning in protein synthesis and destination. Their expression was down-regulated in hairy roots having less lateral branching. This phenotype may have resulted from the manipulation of metabolic activities by the translational machinery.     

Characterization,expression profiling,and functional identification of a gene encoding geranylgeranyl diphosphate synthase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including tanshinone. In this study, a full-length cDNA encoding GGPPS was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmGGPPS (GenBank Accession No. FJ643617). The full-length cDNA of SmGGPPS was 1,234 bp containing a 1,092 bp open reading frame (ORF) encoding a polypeptide of 364 amino acids. Analysis of SmGGPPS genomic DNA revealed that it contained 2 exons and 1 intron. Bioinformatics analyses revealed that the deduced SmGGPPS had extensive homology with other plant GGPPSs contained all 5 conserved domains and functional aspartate-rich motifs of the prenyltransferases. Molecular modeling showed that SmGGPPS is a new GGPPS with a spatial structure similar to other plant GGPPSs. Phylogenetic tree analysis indicated that SmGGPPS belongs to the plant GGPPS super-family and has the closest relationship with GGPPS from Nicotiana attenuate. The functional identification in Escherichia coli showed that SmGGPPS could accelerate the biosynthesis of carotenoid, demonstrating that SmGGPPS encoded a functional protein. Expression pattern analysis implied that SmGGPPS expressed higher in leaves and roots, weaker in stems. The expression of SmGGPPS could be up-regulated by Salicylic acid (SA) in leaves and inhibited by methyl jasmonate (MeJA) in 3 tested tissues, suggesting that SmGGPPS was elicitor-responsive. This work will be helpful to understand more about the role of SmGGPPS involved in the tanshinones biosynthesis pathway and metabolic engineering to improve tanshiones production in S. miltiorrhiza.     

Enhanced secondary metabolite (tanshinone) production of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots in a novel root–bacteria coculture process

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g⁻¹ dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l⁻¹). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.     

Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures

Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.     

Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots

Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Identification of heavy metal-induced genes encoding glutathione S-transferases in the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.     

PEG and ABA Trigger the Burst of Reactive Oxygen Species to Increase Tanshinone Production in Salvia miltiorrhiza Hairy Roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Tanshinones, a group of active compounds in S. miltiorrhiza, are derived from two biosynthetic pathways: the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastids. Water stress is well known to stimulate the accumulation of secondary metabolites in plants. Reactive oxygen species (ROS) serve as important secondary messengers in water stress-induced signal transduction pathways. In this study, the effects of polyethylene glycol (PEG) and abscisic acid (ABA) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of ROS in PEG- and ABA-induced tanshinone production were further elucidated. The results showed that contents and yields of four tanshinones in S. miltiorrhiza hairy roots were significantly enhanced by 2 % PEG and 200?μM ABA. Simultaneously, the mRNA levels and activities of two key enzymes (3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose 5-phosphate synthase) involved in tanshinone biosynthesis were upregulated. Both PEG and ABA were able to trigger the burst of H₂O₂ and O₂ ^?. The PEG- and ABA-induced increases of tanshinone production, gene expression, and enzyme activity were all dramatically suppressed by two ROS scavengers, catalase and superoxide dismutase. In addition, ROS treatments resulted in a significant increase in tanshinone production. These results demonstrated that the MVA and MEP pathways were activated by PEG and ABA to stimulate tanshinone biosynthesis, and the increase of tanshinone production was probably via ROS signaling.     

Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O₂ ⁻ and H₂O₂ were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.