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Biotechnology Advances xxx (2014) xxx–xxx

JBA-06845; No of Pages 12

Contents lists available at ScienceDirect

Biotechnology Advances

j ourna l homepage: www.e lsev ie r .com/ locate /b iotechadv

Research review paper

Metabolic engineering and biotechnological approaches for productionof bioactive diterpene tanshinones in Salvia miltiorrhiza

OFGuoyin Kai a,⁎, Xiaolong Hao a, Lijie Cui a, Xiaoling Ni b, David Zekria b, Jian-Yong Wu c,⁎⁎

a Laboratory of Plant Biotechnology, Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR Chinab Department of General Surgery, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai 200032, Chinac Department of Applied Biology & Chemical Technology, State Key Laboratory of Chinese Medicine and Molecular Pharmacology in Shenzhen, The Hong Kong Polytechnic University, Hung Hom,Kowloon, Hong Kong

⁎ Corresponding author. Tel./fax: +86 21 6432 1291.⁎⁎ Corresponding author. Tel.: +852 3400 8671; fax: +

E-mail addresses: [email protected] (G. Kai), jian-

http://dx.doi.org/10.1016/j.biotechadv.2014.10.0010734-9750/© 2014 Published by Elsevier Inc.

Please cite this article as: Kai G, et al, MetabolSalvia miltiorrhiza, Biotechnol Adv (2014), h

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Keywords:Salvia miltiorrhizaTanshinonesPharmacological activityBiosynthetic pathwayGene cloningMetabolic engineeringHairy root cultureElicitation

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Radix Salviamiltiorrhiza Bunge, generally calledDanshen, is an important Chinese herbwhich ismost effective fortreatment of cardiovascular diseases and inflammations. Tanshinones, a group of abietane diterpenes, represent amajor class of pharmacologically active constituents of Danshen with significant antioxidant, anti-inflammatoryand anti-cancer activities, some of which have been explored as new drug candidates. Biotechnology approacheshave been taken to improve the conventional processes and to develop new processes for efficient production oftanshinones so as to meet the increasing demand. The development of effective biotechnology means requiressufficient understanding of the biosynthetic pathways of tanshinones and themolecular regulationmechanisms.Recently, many genes in the tanshinone biosynthetic pathways have been cloned and functionally identified,which are useful for designing genetic engineering technology for the over production of tanshinones. Trans-formed hairy root culture of S. miltiorrhiza has been one of the most useful experimental systems for metabolicengineering studies and also a potential system for biotechnology production of tanshinones. This reviewsummarizes the chief pharmacological activities and the recent advances in tanshinone biosynthesis andmetabolic regulation, and in the improvement of in vitro production by various biotechnological approaches,and finally gives our views on the future prospects.

© 2014 Published by Elsevier Inc.
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Contents

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Pharmacological activities of tanshinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Cardiovascular protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Antioxidant activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Anti-inflammatory activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Cytotoxic activities and anticancer potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Other pharmacological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Biosynthesis and regulation of tanshinones in S. miltiorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Biosynthesis of tanshinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Cloning and characterization of genes related to tanshinone biosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Genes in the early stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Genes in the middle stage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Genes in the late stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Regulatory mechanisms involved in tanshinone biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Biotechnological approaches to enhancing tanshinone production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Elicitation treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

852 2364 [email protected] (J.-Y. Wu).

ic engineering and biotechnological approaches for production of bioactive diterpene tanshinones inttp://dx.doi.org/10.1016/j.biotechadv.2014.10.001

http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

mailto:[email protected]

mailto:[email protected]


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Endophytic fungi of S. miltiorrhiza. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Production of tanshinones in tissue cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Uncited reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

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Introduction

Salvia miltiorrhiza Bunge (family Labiatae) (Fig. 1) is an importantChinese herbal plant and its dried root, generally named Danshen inChinese or Chinese Red Sage, has been widely used in modern and tra-ditional Chinese medicine (TCM) for the treatment of cardiovascular,and cerebrovascular diseases and various inflammation symptoms(Dong et al., 2011; Wang and Wu, 2010; Xu et al., 2010; Yan, 2013).Danshen has been used either alone or in combination with otherherbs in China and some other countries such as Australia, Germany,and the United States (Cheng, 2007). Raw Danshen roots and formulat-ed Danshen therapeutic products in various dosage forms have beensold in large quantities in China, among which the Fufang DanshenDripping Pill and FufangDanshen Tablet are themostwidely distributed(Zhou et al., 2005). The Fufang Danshen Dripping Pill which is listed inthe official China pharmacopeia has been widely used in China, andalso in several other countries such as Russia, Cuba, the Korean Republic,Saudi Arabia and Vietnam (Zhang et al., 2012; Zhou et al., 2005). It isalso the first TCM drug approved for clinical trials in the USA by theFood and Drug Administration (FDA), passed phase II trials in 2010,and approved for phase III clinical trials in 2012 on patientswith chronicstable angina pectoris (http://clinicaltrials.gov/, No. NCT00797953). Themarket value of Fufang Danshen Dripping Pill in China was over US$320 million in 2013, accounting for 15% of all cardiovascular drugs(www.tasly.com).

As a major class of pharmaceutically bioactive constituents ofDanshen, tanshinones are a group of more than 40 abietane diterpenessuch as tanshinone I, tanshinone IIA, tanshinone VI, dihydrotanshinone,and cryptotanshinone (Fig. 2) (Dong et al., 2011). Some of thesetanshinones have exhibited various pharmacological activities, such asanti-oxidative, anti-inflammatory, anti-proliferative, antibacterial andanti-tumor properties (Gao et al., 2012; Gong et al., 2011; Park et al.,

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Fig. 1. Live Salvia miltiorrhiza flowers (left) and roots (right)

Please cite this article as: Kai G, et al, Metabolic engineering and biotechnolSalvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biot

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2009; Xu et al., 2010; Zhang et al., 2012), and have great potential forclinical application. Tanshinone IIA is one of the most active speciesand its derivative, sodium tanshinone IIA sulfate (STS), has been widelyevaluated for treatment of cardiovascular diseases (CVD), cancer, hepat-ic fibrosis, and neurodegenerative diseases (Cheng, 2007; Takahashiet al, 2002;Wei et al., 2014). The proven therapeutic effects of Danshenand the rising public interest in herbal medicine have driven theincreasing demand for the Danshen herb. Because of the scarce andextinct natural species, the commercial supply of Danshen for herbalmedicine and preparation of various extracts has been mainly reliedon field or domestic cultivation of the S. miltiorrhiza plants. Danshenroots on the market have been mainly produced on the agriculturefarms in several Chinese provinces such as Shandong, Henan, Shanxi,Anhui, Sichuan and Hebei. The annual consumption of Danshen hasexceeded 20 million kilograms (at US$2–3 per kg) in China (www.gshsyy.com). However, field cultivation of herbal plants has severaldisadvantages including the slow plant growth and long productionperiod, high labor cost, occupation of farm land, and contamination byfertilizers and pesticides (Hao et al., 2014; Kai et al., 2011a). Becauseof the low contents of tanshinones in the field-cultivated plant roots,large amount of roots is required for the extraction and isolation oftanshinones for treatment uses (Kai et al., 2010; Liao et al., 2009). There-fore, it is of significance to develop new and more effective alternativesto the conventional processes for tanshinone production.

Two of the most widely explored processes for efficient productionof tanshinones andmanyother bioactive phytochemicals include chem-ical synthesis and enhanced biosynthesis through biotechnologyapproaches (Dong et al., 2011; Dreger et al., 2010; Kai et al., 2011a;Wang et al., 2010). Although various strategies have been developedfor total organic synthesis of tanshinones (Chang et al., 1990;Danheiser et al., 1995;Wang et al., 2004), the synthetic routes are ratherlong, involving numerous reaction steps with very low final yields.

after plantation in an experimental field for two years.

ogical approaches for production of bioactive diterpene tanshinones inechadv.2014.10.001

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Fig. 2. The chemical structures of four major tanshinone compounds.

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Environmental impact is another major concern with the chemicalmethods. Therefore, it is not commercially feasible to obtain tanshinonesvia chemical synthesis. Therefore, various biotechnology approacheshave been explored for enhancing the biosynthesis and production oftanshinones, most of which have been carried out in or through planthairy root cultures. Although some endophytic fungi isolated fromS. miltiorrhiza have been reported to produce tanshinones, the amountsare much lower than in the host plant (Ming et al., 2012).

With the rapid advancement and wide application of plant biotech-nology, metabolic engineering has become a feasible alternative forimproving the production of targeted metabolites. Progress in this di-rection has been made for production of tanshinones by manipulationof the key biosynthetic genes in the S. miltiorrhiza genome using trans-formed hairy roots, and large scale culture of S. miltiorrhiza hairy rootsor regeneration of transgenic plants (Hao et al., 2014; Kai et al., 2011a;Shi et al., 2014). This review is to summarize the recent advancesin the understanding of tanshinone biosynthetic pathways, metabolicregulations, and various biotechnology approaches for more efficientproduction of tanshinones, and the future prospects.

Pharmacological activities of tanshinones

Danshen and its related products have been widely used in clinicalpractice in different forms such as tablets, capsules, granules or liquidformula (Zhou et al., 2005). As a major class of bioactive constituentsin Danshen, tanshinones may be attributable to the therapeutic effects.Numerous studies havedemonstrated various bioactivities of tanshinonessuch as antioxidant, anti-inflammatory, anticancer, and antibacterialactivities, and protection on the cardiovascular system (Gao et al., 2012;Park et al., 2009; Xu et al., 2010; Yan, 2013; Zhang et al., 2012).

Cardiovascular protection

Tanshinones have been tested in various model systems for thetreatment of cardiovascular diseases, such as coronary heart disease, hy-perlipidemia, and cerebrovascular diseases (Gao et al., 2012; Park et al.,2009; Xu et al., 2010; Yan, 2013). Among the numerous tanshinones,


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tanshinone IIA has been most widely studied and shown to preventatherogenesis, cardiac injury and hypertrophy (Gao et al., 2012).Tanshinone IIA could significantly reduce the size of myocardial infarct,which may be related to its free radical scavenger property in themyocardial mitochondrial membrane (Zhou et al., 2005). TanshinoneIIA also inhibited the oxidation of low-density lipoproteins (LDL),alter monocyte adhesion to the endothelium, modulate migration andproliferation of smooth muscle cell, reduce macrophage cholesterolaccumulation, and reduce proinflammatory cytokine expression andplatelet aggregation (Gao et al., 2012; Liu et al., 2013).

Tanshinone VI can suppress hypertrophy of cardiomyocytes inneonatal rat hearts induced by the insulin-like growth factor-1 (IGF-1)via the attenuation of extracellular signal-regulated kinase 1/2 (ERK)activation (Kawahara et al., 2004). [3H]-leucine incorporation into IGF-1-untreated cells was unaltered when the cells were incubated in thepresence of tanshinoneVI (0.1 to 10 μM),while IGF-1-induced increasesin [3H]-leucine incorporation into the trichloroacetic acid (TCA)-insolublefraction andphosphorylated ERKwere attenuatedwith 10 μMtanshinoneVI. It appeared that tanshinone VI did not affect protein synthesis inmyo-cardium, but attenuatedmyocardial hypertrophy induced by IGF-1. Thesefindings suggest the potential of tanshinone VI as a drug candidate forimproving cardiac remodeling during the development of heart failure.

Antioxidant activities

PF2401-SF, a partially purified extract of Danshen, and itsmain com-ponents, tanshinone I, tanshinone IIA and cryptotanshinone, can protectagainst liver toxicity in vivo and in vitro due to its antioxidant potential(Park et al., 2009). They can also protect against acute and sub-acuteliver damage induced by carbon tetrachloride, and protect primarycultured rat hepatocytes from tertiary-butyl hydroperoxide (tBH) orD-galactosamine (GalN). In addition, tanshinone I (10 and 40 μM),tanshinone IIA (40 μM), and cryptotanshinone (40 μM) could inhibitlactate dehydrogenase leakage, glutathione (GSH) depletion, lipidperoxidation and free radical generation in vitro. PF2401-SF fortifiedwith tanshinone I, tanshinone IIA, and cryptotanshinone showedprotective effects on rat liver at a lower dose than the ethanol extractof S. miltiorrhiza due probably to its antioxidant effects (Park et al.,2009).

Anti-inflammatory activity

Tanshinone IIA has shown anti-inflammatory activity by inhibitingthe production of pro-inflammatory mediators in murine macrophageRAW264.7 cells stimulated with lipopolysaccharide (LPS) via threedifferent pathways (Fan et al., 2009). Particularly, tanshinone IIAinhibited LPS-induced IκBα degradation and nuclear factor κB (NF-κB)activation through suppression of the NF-κB-induced kinase–IκBαkinase (NIK–IKK) pathway, the mitogen-activated protein kinases(MAPKs) pathway such as the p38 MAPK (p38) pathway, the extracel-lular signal-regulated kinases 1/2 (ERK1/2) pathway, and the c-Jun N-terminal kinase (JNK) pathway (Jang et al., 2006). Tanshinone IIA alsoinhibited LPS-induced nitric oxide synthase (iNOS) gene expressionand nitric oxide (NO) production of the RAW264.7 cells and the expres-sion of inflammatory cytokines (IL-1β, IL-6, and TNF-α) (Fan et al.,2009). These findings suggest the potential of tanshinone IIA as a selec-tive estrogen receptor modulator (SERM) useful for the treatmentof inflammation-associated neurodegenerative and cardiovasculardiseases without increasing the risk of breast cancer (Fan et al., 2009).

Antibacterial activity

Cryptotanshinone and dihydrotanshinone I have shown antibacterialactivity against a number of Gram positive bacteria including Bacillussubtilis (Lee et al., 1999). A recombination-deficient mutant strain ofB. subtiliswas more sensitive to the two tanshinones, exhibited reduced



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hypersensitivity in the presence of an antioxidant such as dithiothreitol(Lee et al., 1999). However, the two tanshinones showed nonselectiveinhibitory action on DNA, RNA, and protein synthesis in B. subtilis (Leeet al., 1999). It has been suggested that superoxide radical formation isa major cause for the antibacterial action of cryptotanshinone anddihydrotanshinone I (Lee et al., 1999). Another study has demonstratedthat cryptotanshinone showed significant antibacterial activity againsttwenty one Staphylococcus aureus strains (Feng et al., 2009). The antibac-terial action of cryptotanshinonemay be related to its action as an activeoxygen radical generator through transcriptome analysis, creating anoxygen-limiting state in the bacteria.

Cytotoxic activities and anticancer potential

Numerous recently studies have evaluated the potential anti-tumoreffects of tanshinones, mainly by in vitro cytotoxicity assays on variouscancer cells such as lung, breast, prostate, colon, liver, kidney, stomach,ovary, cervix, glioma cancer cells, and melanoma, rhabdomyosarcomaand leukemia cells (Table 1). Several possible mechanisms of actionhave been postulated for the cytotoxic activities of tanshinones, suchas anti-proliferation, pro-apoptosis, anti-angiogenesis, induction ofdifferentiation, and inhibition of adhesion, migration, invasion andmetastasis. Tanshinones may also exert the cancer cell inhibitory activ-ities bymodulating the inflammatory and immune responses, inhibitingtelomerase, interacting with the DNA minor groove and activating p53tumor suppressor, or regulating specific pathways such as the androgenreceptor (AR) or signal transducer and activator of transcription(STAT3) (Liu et al., 2013; Zhang et al., 2012). Of the three testedtanshinone compounds (CT, T-IIA and T-I), tanshione I showed themost potent inhibiting activities on prostate cancer in vitro and inmice, and Aurora A kinase was identified as a possible target of thetanshinone action (Gong et al., 2011). With the notable cytotoxic activ-ities of tanshinones on various cancer cells, the potential anticancer

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Table 1Cytotoxic activities of tanshinones on cancer cells for anticancer.

Tanshinone Anticancer

Tanshinone I Lung cancerBreast cancerProstate cancerLeukemiaColon cancer

Tanshinone IIA Colon cancer

Breast cancer

Liver cancer

Kidney cancerStomach cancerOvarian cancerLung cancer

Prostate cancerCervical cancerLeukemia

Glioma cancerCryptotanshinone Leukemia

MelanomaRhabdo-myosarcoma

Dihydrotanshinone I Colon cancerLiver cancer

15,16-Dihydrotanshinone I Prostate cancerBreast cancer


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effects can be further examined in various animalmodels and by clinicalhuman trials before further development and application of anti-tumoragents from the tanshinones.

Other pharmacological activities

Tanshinone I has been found to ameliorate the learning andmemoryimpairments in mice (Kim et al., 2009). Firstly, tanshinone I increasedthe latency time versus a vehicle-treated control group in the passiveavoidance task of the test animals (Kim et al., 2009). Western blotanalysis and immunohistochemical assays showed that tanshinone Iincreased the levels of phosphorylated cAMP response element bindingprotein (pCREB) and phosphorylated extracellular signal-regulated ki-nase (pERK) in the hippocampus (Kim et al., 2009).Moreover, thewest-ern blot analysis showed that tanshinone I reversed the diazepam- andMK-801-induced inhibition of ERK and CREB activation in hippocampaltissues (Kim et al., 2009). The study suggests the potential of tanshinoneI as a drug candidate for cognitive deficits.

Dihydrotanshinone and cryptotanshinone have been shown to inhibitthe acetylcholinesterase (AChE) activity in a dose-dependent mannerwith 50% inhibitory concentration (IC50) values of 1.0 μM and 7.0 μM, re-spectively (Ren et al., 2004). Therefore, these tanshinone compoundshave the potential for application as a new drug to treat Alzheimer's dis-ease (Dong et al., 2011; Ren et al., 2004). Furthermore, the lipo-hydro par-tition coefficient (clogP) values of dihydrotanshinone, cryptotanshinone,tanshinone I and tanshinone IIA were 2.4, 3.4, 4.8 and 5.8, respectively,suggesting their ability to penetrate the blood–brain barrier (Ren et al.,2004).

Although the pharmacological activities of tanshinones have beenextensively evaluated, the mechanisms of action are not well under-stood. Moreover, the low aqueous solubility and poor membranepermeability of tashinones need to be overcome for their therapeuticapplications.

Cell line Reference

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Liu et al., 2013bSu et al., 2008b

BT-20MCF-7, MDA-MB-231

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J5; BEL-7402HepG2, Hep3BPLC/PRF/5SMMC-7721

Chien et al., 2012Dai et al., 2012Lee et al., (2010)Yuan et al., (2004)

786-O Wei et al., (2012)MKN45, SGC7901 Chen et al., (2012)COC1/DDP Jiao et al., (2011)H146A549

Cheng et al., 2010Chiu et al., 2010

LNCaP Won et al., (2012)HeLa Pan et al., (2010)NB4 and MR2THP-1T-cell

Zhang et al., (2010)Liu et al., (2009)

U251 Wang et al., (2007)K562 Ge et al., (2012)mice B16/B16BL6 Chen et al., (2011)Rh30 Chen et al., (2010)HCT116 and Caco-2 Wang et al., (2013)HepG2 Lee et al., (2009)DU145 Chuang et al., (2011)MCF-7, MDA-MB-231 Tsai et al., (2007)



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Biosynthesis and regulation of tanshinones in S. miltiorrhiza

Biosynthesis of tanshinones

Tanshinones belong to diterpenes which are biosynthesized fromtwo major five-carbon intermediates, isopentenyl diphosphate (IPP)and its isomer dimethylallyl diphosphate (DMAPP), which can be con-verted to one another with the catalysis of isopentenyl diphosphateisomerase (IPI). The tanshinone biosynthesis process, which is still notfully characterized, can generally be divided into three stages, startingfrom the formation of a universal five-carbon precursor IPP, followedby the formation of a key 20-carbon intermediate GGPP, and endingwith the formation of the target product tanshinones via the late specificpathways as shown in Fig. 3.

In the early stage, there are two distinct routes responsible for thesynthesis of universal IPP in higher plants, the well-known mevalonate

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Fig. 3. The biosynthetic pathways of tanshinones in S.miltiorrhiza. The solid arrows indicate knpathways: AACT, acetyl-CoA C-acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthphosphomevalonate kinase; MDC, mevalonate 5-diphosphate decarboxylase; DXS, 1-deoxy-D-xyl2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase; CMK, 4-(cytidine 5-diphospho)-2-C-m1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase; HDR, 1-hydroxy-2-methyl-2-(E)-geranylgeranyl diphosphate synthase; CPS, copalyl diphosphate synthase; KSL, kaurene synthase-


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(MVA) pathway in the cytoplasm and the more recently discoverednon-MVA 1-deoxyxylulose 5-phosphate (DXP) pathway (also called 2-C-methyl-D-erythritol-4-phosphate pathway, MEP pathway) in plastid(Lange et al., 2000; Liao et al., 2009; Yan et al., 2009; Zhang et al.,2011). Six catalytic enzymes are involved in theMVA pathway includingacetyl-CoAC-acetyltransferase (AACT), 3-hydroxy-3-methylglutaryl-CoAsynthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR),mevalonate kinase (MK), phosphomevalonate kinase (PMK) andmevalonate 5-diphosphate decarboxylase (MDC) and, seven en-zymes are involved in the DXP pathway including 1-deoxy-D-xylulose5-phosphate synthase (DXS), 1-deoxy-D-xylulose5-phosphatereductoisomerase (DXR), MEP cytidylyltransferase (MCT), 4-(cytidine5-diphospho)-2-C-methylerythritol kinase (CMK), 2-C-methylerythritol2,4-cyclodiphosphate synthase (MECPS), hydroxymethybutenyl 4-diphosphate synthase (HDS) and hydroxymethylbutenyl 4-diphosphatereductase (HDR) (Liao et al., 2009; Shi et al., 2014; Wang and Wu,

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own steps and the dashed arrows represent unknown steps. Enzymes in the biosyntheticase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase;MK,mevalonate kinase; PMK, 5-ulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphatereductoisomerase; MCT,ethyl-Derythritolkinase; MECPS, 2-C-methylerythritol 2,4-cyclodiphosphatesynthase; HDS,butenyl-4-diphosphate reductase; IPPI, isopentenyl-diphosphate delta-isomerase; GGPPS,like; CYP76AH1, cytochrome P450 enzyme (CYP) 76AH1.



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2010; Xu et al., 2010; Yan et al., 2009; Zhang et al., 2011). Although theMVA and DXP pathways exist in different compartments, the two path-ways are interrelated and can cross talk to provide basic precursors fortanshinone biosynthesis (Ge and Wu, 2005a; Kai et al., 2011; Shi et al.,2014).

In themiddle stage, the key 20-carbon intermediate GGPP is derivedfrom the universal five-carbon precursor catalyzed by geranyl diphos-phate synthase (GPPS), farnesyl diphosphate synthase (FPPS) andgeranylgeranyl diphosphate synthase (GGPPS). GPPS catalyzes the reac-tion of IPP andDMAPP to form10-carbon geranyl pyrophosphate (GPP),after which FPPS catalyzes GPP and IPP to form 15-carbon farnesyldiphosphate (FPP), and then 20-carbon GGPP is synthesized from onemolecule of FPP and the third molecule of IPP by GGPPS (Kai et al.,2010; Xu et al., 2010).

The last stage involves the formation of various terpenoids under thecatalysis of terpene synthases/cylases (TPSs), such as copalyl diphos-phate synthase (CPS), kaurene synthase (KS), miltiradiene oxidaseCYP76AH1 and other modifying enzymes (Ma et al., 2012). GGPP canbe cyclized to form normal copalyl diphosphate (CPP) by copalyldiphosphate synthase (CPS), and the normal CPP catalyzed into anabietane-type diterpenoid named miltiradiene by kaurene synthase-like (KSL) through further cyclization and rearrangement (Gao et al.,2009; Xu et al., 2010). Subsequently, miltiradiene is converted toferruginol by miltiradiene oxidase CYP76AH1 (Guo et al., 2013), andthen to the target tanshinone products by others enzymes.

Cloning and characterization of genes related to tanshinone biosynthesis

With increasing therapeutic applications of Danshen and recognitionof the bioactivities of tanshinones in recent years, considerable researchefforts have been made to understand biosynthesis of tanshinones inS. miltiorrhiza at the molecular level. Recently several genes involved inthe biosynthetic pathways of tanshinones have been successively clonedfrom S.miltiorrhiza by various approaches as described below.

Genes in the early stageAACT is the first enzyme in the MVA biosynthesis pathway, catalyz-

ing two molecules of acetyl-CoA to acetoacetyl-CoA (Igual et al., 1992).One SmAACT cDNA (EF635969), which is comprised of a 1200-bp openreading frame (ORF) encoding a 399 amino acid protein, was clonedfrom S. miltiorrhiza hairy roots using a cDNA microarray and rapid am-plification of cDNA ends (RACE) strategy (Cui et al., 2010). SmAACTexpressed in roots, stems and leaves with higher level in the formerthan the other two tissues and its expression was up-regulated byboth yeast extract (YE) and Ag+ elicitors as detected by cDNA microar-ray and quantitative RT-PCR (reverse transcription-polymerase chainreaction) techniques (Cui et al., 2010).

HMGS catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form HMG-CoA as an early step in the MVA pathway (Kai et al.,2006; Nagegowda et al., 2004). A new full-length SmHMGS cDNA(FJ785326 in GenBank) was isolated by RACE containing a 1381-bpORF encoding a 460 amino acid protein (Zhang et al., 2011). Expressionprofile analysis showed that SmHMGS was constitutively expressedin leaves, stems and roots, and was also up-regulated in response toexogenous plant hormone including salicylic acid (SA) and methyljasmonate (MJ) (Zhang et al., 2011).

HMGR conversion of 3-hydroxy-methylglutaryl-CoA (HMG-CoA) toMVA has been identified as the first key step in the MVA pathway inplants (Bach et al., 1995; Jiang et al., 2006). A full-length cDNA ofSmHMGR (EU680958 in GenBank), a rate-limiting enzyme of the MVApathway, has been isolated from S. miltiorrhiza by RACE by our group,which contained a 1695-bp ORF encoding a 565 amino acid protein.Expression analysis in tissues has revealed that SmHMGR is a constitu-tively expressed gene with different levels in various tissues such asroots (high), stems (moderate) and leaves (weak). The expression ofSmHMGR was up-regulated in response to exogenous SA and MJ (Liao


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et al., 2009). Another full-length cDNA of SmHMGR2 (FJ747636 inGenBank) has been cloned by RACE with a 1653-bp ORF (Dai et al.,2011). SmHMGR2 was strongly expressed in leaf, stem and root tissuesand could functionally complement the yeast HMGR mutant JRY2394.

DXS catalyzes pyruvate and glyceraldehyde 3-phosphate to form 1-deoxy-D-xylulose 5-phosphate, which is known as the first step in theDXP pathway in plants (Estévez et al., 2000; Floß et al., 2008). Twofull-length cDNAs encoding 1-deoxy-D-xylulose 5-phosphate synthase(SmDXS1 and SmDXS2) were first cloned from S. miltiorrhiza by RACEin our team. The full-length cDNA of SmDXS1 (EU670744.1 in GenBank)was 2519 bp containing a 2145-bp ORF encoding 714 amino acidsand the full-length cDNA of SmDXS2 (FJ643618.1 in GenBank) was2522 bp containing a 2175-bp ORF encoding 724 amino acids. SmDXS1was expressed in all tested tissues including roots, stems, and leaves,whereas SmDXS2 was expressed only in roots, suggesting that it ismainly involved in tanshinone biosynthesis (Kai et al., unpublisheddata), which is consistent with the results reported recently by Yanget al. (2013). Some other DXS members have also been found usinggenome-wide identification methods (Ma et al., 2012).

DXR, an enzyme responsible for conversion of DXP to MEP, the sec-ond step of the DXP pathway, may play an important role in regulatingthe DXP pathway (Carretero-Paulet et al., 2002; Lois et al., 2000). TheSmDXR gene (FJ476255 in GenBank) has been isolated and character-ized in S. miltiorrhiza (Yan et al., 2009). Expression profile analysisshowed that SmDXR was constitutively expressed in leaves, stems androots, and was responsive to elicitors such as MJ and SA (Yan et al.,2009). Wu et al (2009) also cloned a SmDXR and verified its functionwith the complementation of an Escherichia coli dxr mutant.

CMK is a middle enzyme in the DXP pathway, and is the only kinaseof the DXP pathway. A CMK gene (EF534309 in GenBank) was isolatedfrom hairy roots of S. miltiorrhiza and had a 1493-bp cDNA includingan 1191-bp ORF encoding a protein of 396 amino acids. The pI of de-duced protein was 6.78 and its calculated molecular weight was about43 kDa, which was similar to other reported plant CMKs (Wang et al.,2008). The expression of SmCMK and accumulation of tanshinoneswere both increased by MJ stimulation (Wang et al., 2008).

HDR is a terminal enzyme in the DXP pathway (Hsieh et al., 2005). Afull-length cDNA of SmHDR (JX233817 in GenBank) has been isolatedfrom S. miltiorrhiza hairy roots, which consists of 1647 nucleotideswith a 1392-bp ORF encoding a 463 amino acid protein (Cheng et al.,2013; Hsieh et al., 2005). The SmHDR expression appeared to be posi-tively correlated to tanshinone accumulation in S. miltiorrhiza whentreated by Ag+ (Cheng et al., 2013). Hao et al. (2013) have isolatedanother full-length cDNA of SmHDR from S. miltiorrhiza (JX516088 inGenBank) and identified its function in E. coli. Transcription analysisrevealed that expression of SmHDR1 was high in leaves and low inroots and stems. In hairy root cultures of S. miltiorrhiza, SmHDR1 wasresponsive to MJ and SA, but not abscisic acid (ABA) (Hao et al., 2013).

Genes in the middle stageIPPI is an important enzyme in the terpenoid biosynthetic pathway

(Cui et al., 2011). An IPPI gene SmIPPI with high homology with otherplant IPIs has been screened out by analyzing transcriptome sequencesof S. miltiorrhiza (Yang et al., 2011). The putative SmIPPI consisted of1243 nucleotides, including a 681-bp ORF and encoding a 226 aminoacid protein. It has been found by qRT-PCR analysis that SmIPPI wasexpressed in different developmental stages and organs, and could beinduced byMJ and a fungal elicitor (Yang et al., 2011). With microarraysome genes involved in tanshinone biosynthesis have been isolatedand characterized, including isopentenyl diphosphate isomerase 2(SmIPPI2) and farnesyl diphosphate synthase (SmFPS) (Cui et al., 2011).

A full-length cDNA of SmGGPPSwith 1234 bp has been isolated fromS. miltiorrhiza by RACE (FJ643617 in GenBank), which comprised a1092-bp ORF and encoded a 364 amino acid protein (Kai et al., 2010).SmGGPPS accelerated the biosynthesis of carotenoid in geneticallyengineered E. coli, suggesting that SmGGPPS was a functional protein.



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Expression profile analysis results revealed that SmGGPPSwas constitu-tively expressed in the tested tissues including leaves, stems and roots,and was stimulated by SA but suppressed by MJ (Kai et al., 2010).

Genes in the late stageA full-length cDNA of CPS containing a 2382 bp ORF has been isolated

from S.miltiorrhiza hairy root by RACE (Gao et al., 2009). The peptide hasbeen deduced to be composed of 793 amino acids with a pI of 6.45 and amolecular weight of 90.36 kDa. After MJ elicitation, the expression ofSmCPS was strongly stimulated as well as the contents of tanshinoneIIA and cryptotanshinone.

The kaurene synthase-like (KSL) gene located downstream of SmCPShas also been cloned from S. miltiorrhiza hairy root by RACE (Gao et al.,2009). The 2110 bp full-length cDNA of SmKSL containing 1788 bp ORFwas obtained, which encoded a 595 amino acid protein, and the molecu-larweight andpIwere estimated to be68.3 kDa and6.0, respectively (Gaoet al., 2009). A positive correlation was observed between SmKSL expres-sion and the accumulation of tanshinone IIA and cryptotanshinone inS.miltiorrhiza hairy roots subjected to MJ treatment (Gao et al., 2009).

Recently, by a stable isotope labeling method and next-generationsequencing approach, six candidate Cytochrome P450 enzymes (CYPs)genes have been found to be co-regulated with the diterpene synthasegenes in both the rhizomes and hairy roots of S. miltiorrhiza (Guo et al,2013). CYP76AH1, one of six CYP genes, has been successfully demon-strated to catalyze a unique oxidation cascade on miltiradiene to pro-duce ferruginol both in vitro and in vivo (Guo et al., 2013). Based onpreviousmiltiradiene-producing Saccharomyces cerevisiae, introductionof CYP76AH1 led to heterologous production of ferruginol at 10.5 mg/Lin yeast. This is of significance for further elucidating the biosynthesisof tanshinones and for the production of terpenoids in the constructedmicrobial cell factories in the future.

Regulatory mechanisms involved in tanshinone biosynthesis

Although significant progress has beenmade in gene isolation, thereis limited information about the key gene targets, the catalytic enzymesinvolved in the late stage, and the underlying molecular regulationmechanisms in the tanshinone biosynthetic pathway of S. miltiorrhiza.Knowledge of the key gene targets is essential for development of met-abolic engineering strategies for improving the yield of tanshinones. Re-cently, we have explored the strategy of using differential expressionprofile combined with elicitation in S. miltiorrhiza hairy roots to screenthe key genes in tanshinone biosynthesis (Kai et al., 2012a), and appliedthese genes for metabolic engineering of the S. miltiorrhiza hairy roots(Kai et al., 2011a; Shi et al., 2014).

Most of the above genes involved in tanshinone biosynthesis havebeen found responsive (at the transcription level) to elicitors suchas MJ and YE, concurring with their induction effects on tanshinoneproduction as reviewed below. Based on the literature data, it may bededuced that the tanshinone biosynthesis pathway is globally up-regulated by certain transcription factors. The cloning of the tanshinonebiosynthetic genes enables the isolation and analysis of the upstreamregulation sequence, whichwill be helpful to understand the regulationof the gene expression and the molecular induction mechanisms forfurther improvement of tanshinone production. However, to our bestknowledge, there are no published studies on transcription factors in-volved in tanshinone biosynthesis. Therefore, isolation and functionalanalyses of global regulatory factors on tanshinone biosynthesis aswell as the regulatory mechanisms remain to be further investigated.Application of the most recent transcriptome sequencing technologiesto S. miltiorrhiza (Hua et al., 2011; Yang et al., 2013; Kai et al., unpub-lished data) may greatly facilitate the isolation and characterization ofrelated genes and transcription factors. Isolation and screening of thegenes related to tanshinone biosynthesis is essential for developmentand application of biotechnological approaches for the enhancementof tanshinone production.


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Biotechnological approaches to enhancing tanshinone production

Danshen as well as most other herbs used in traditional Chinesemedicine ismainly produced byfield cultivation of themedicinal plants.Field cultivation of medicinal plants is a time-consuming and labor-intensive process, from which the yield and property of herb crop isoften affected by environmental factors such as soil, climate, pathogensand pests. Field cultivated herb crops are also prone to variation in qual-ity and content of active ingredients and to contamination of fertilizers,pesticides and heavy metals. Application of plant biotechnology isamong the most attractive and effective measures for overcomingthese problems and achieving efficient and sustainable production ofmedicinal plants and their bioactive constituents. This part is on the bio-technological approaches for enhanced production of tanshinones withthe cell and hairy root cultures and endophytic fungi derived fromS. miltiorrhiza.

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OMetabolic engineering

With the successful isolation of several genes involved in thetanshinone biosynthesis pathways in recent years, it is more feasibleto apply metabolic engineering for improving tanshinone productionin the transformed hairy roots (Kai et al., 2011a; Shi et al., 2014).Through a push–pull strategy, the production of tanshinones has beensignificantly enhanced by up to 4.74-fold in a transgenic line HG9 co-expressing of SmHMGR and SmGGPPS in comparison with the controland a single-gene over-expression hairy root line (Kai et al., 2011a).Meanwhile, co-expression of SmHMGR and SmDXR has also increasedtanshinone production in S.miltiorrhiza hairy root lines, and further im-proved after elicitation by yeast extract and/or Ag+ as reported recently(Shi et al., 2014). The hairy root culture derived from infection ofthe plant tissues by Agrobacterium rhizogenes bacterium carryingthe Ri T-DNA plasmid has been one of themost useful systems for intro-ducing foreign genes into the host plant. Hairy root cultures have beenwidely used as potential production systems for plant secondarymetab-olites and also convenient experimental systems for studying themeta-bolic processes and physiological responses to pathogens, stress andelicitors under well-controlled conditions (Li et al., 2008; Georgievet al., 2012; Kai et al., 2012b; Hao et al., 2014).

Compared to the extraction from natural plant sources and the pro-duction of secondary metabolites in plant tissue cultures, it may bemore efficient to produce the desired metabolites by geneticallyengineered microbial cells. Based on analysis of substrate availability,metabolic flux and the energetics of resource utilization for isoprenoidbiosynthesis in genetically engineered yeast S. cerevisiae with endoge-nous deregulated erg9 and/or coq1 gene, Huang et al. (2013) foundthat isoprenoid biosynthesis was closely related to mitochondrial func-tion. With the construction of a miltiradiene synthetic pathway inS. cerevisiae using a pathway engineering strategy, four consecutivegenes including SmCPS, SmKSL, GGPPs and FPS (farnesyl diphosphatesynthase) have been fused and introduced into the yeast cell (Zhouet al., 2012). The miltiradiene production was significantly enhancedin the engineered yeast cell lines, e.g. to a maximal yield of 365 mg/Lin a 15 L bioreactor with the YJ2X strain, whereas byproduct accumula-tion was reduced (Zhou et al., 2012). This study has demonstrated thepromising potential for producing the target metabolites (tanshinones)by successively stacking the specific genes involved in the late stage ofthe tanshinone biosynthesis pathway into genetically modified yeast.

Metabolic engineering in S. miltiorrhiza plant and microbial cellfactories is expected to make new breakthroughs for enhancedtanshinone production. However, it requires sufficient understandingof the tanshinone biosynthesis pathways and the genetic regulationmechanisms. Moreover, the synthetic biology strategy is a promisingnew and powerful tool for enhancing tanshinone production in plantor microbial cell factories, as envisaged from the successful, high-yield



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production of artemisinic acid, a precursor of artemisinin, in geneticallymodified yeast strains (Paddon et al., 2013).

Elicitation treatment

Elicitation, the treatment of plant cells with biotic and abiotic elici-tors, is one of the most common and effective strategies for stimulatingsecondary metabolite production in plant tissue cultures (Wang andWu, 2013). Elicitor is a term originated from plant science which gener-ally refers to the agents or stimuli that can induce phytoalexin synthesisand defense responses in the challenged host plant. Therefore, theefficacy of elicitation is on the basis that the accumulation of mostsecondary metabolites in plants is part of the defense responses ofplants to pathogen infection and environmental stresses. A variety ofbiotic or abiotic elicitors have been used to enhance the tanshinoneproduction in S. miltiorrhiza hairy root and cell cultures, such as yeastextract (YE), bacterial extract and culture broth, β-aminobutyric acid(BABA), silver (Ag+), cadmium (Cd+), sodium nitroprusside (SNP),polyethylene glycol (PEG) and abscisic acid (ABA) (Table 2).

In the early studies by Chen et al. (2000a; 2000b); Chen et al. (2001),a yeast elicitor (YE) prepared by ethanol precipitation of yeast extractaqueous solution (composed mainly of carbohydrate polymers withprotein) was applied to induce and stimulate the accumulation ofsecondaymetabolites (tanshinone and phenolic acids) in liquid culturesof S. miltiorrhiza hairy roots and transformed cells. YE has then beenused as an effective elicitor in several later studies, separately or in com-binationwith other elicitors, to enhance the tanshinone prodcution in S.miltiorrhiza hairy root and cell cultures (Wang and Wu, 2010; 2013).The non-protein amino acid, β-aminobutyric acid (BABA), havingan important role in activating the defense response of plants againstpathogens (Jakab et al., 2001),was able to increase the tanshinone accu-mulation in S. miltiorrhiza hairy roots up to 4.5-fold when used alone,and up to 9.4-fold when combined with YE (Ge and Wu, 2005b). Itwas suggested that BABA potentiated the elicitation of S. miltiorrhizahairy roots by YE, leading to more significant enhancement oftanshinone production than each used alone. In other studies, YE wasapplied in combination with the silver ion Ag+ to the S. miltiorrhizahairy root culture leading to more significant increase of tanshinoneproduction (Ge and Wu, 2005a; Kai et al., 2012a). Based on specificpathway inhibitor experiments, tanshinone accumulation was suggestto undergo through the DXP pathway mainly as inferred by Ag+ andYE treatment (Ge and Wu, 2005a), which was further confirmed bytransgenic experiment in our group (Kai et al., 2011a). The combinationof elicitor treatment with effective process strategies, YE, in situ adsorp-tion and recovery of tanshinones with a hydrophobic polymeric resin,and periodic replenishment of fresh medium, led to more dramaticincrease in the tanshinione production (Yan et al., 2005).

Bacillus cereus, a plant growth-promoting rhizobacterium, was inoc-ulated into the S. miltiorrhiza hairy root culture to form a root-bacterialco-culture system, in which the total tanshinone content of roots was

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Table 2Elicitors applied for stimulation of tanshinone accumulation in S. miltiorrhiza hairy root and ce

Elicitora Culture system

YE Hairy rootAg+ Hairy rootBABA, BABA + YE Hairy rootAg+,YE Hairy rootBacillus cereus bacteria Hairy rootOsmotic stress (sorbitol) Hairy rootHeavy metal ions, polysaccharides from Bacillus cereus,SA,MJ, osmotic stress (sorbitol)

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MJ, SNP Hairy rootPEG, ABA and MJ Hairy root

a ABA, abscisic acid; BABA, β-aminobutyric acid; MJ, methyl jasmonate; PEG, polyethylene g


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increased by more than 12-fold compared with the control culture(Wu et al., 2007). The study demonstrated an effective hairy root–bacterium co-culture for improving the production of secondarymetab-olites in hairy root cultures. The enhanced tanshinone production in thehairy root–bacteria co-culture system was attributed to the elicitorcompounds such as polysaccharides released from the bacteria (Wuet al., 2007). The polysaccharide fraction of an endophytic fungusTrichoderma atroviride isolated from the root of S. miltiorrhiza has alsobeen shown to be an effective elicitor for stimulating the tanshinoneaccumulation and the growth of S. miltiorrhiza hairy roots as well asthe transcription of genes in the tanshinone biosynthesis pathway(Ming et al., 2013).

In S. miltiorrhiza cell culture the effect of several elicitors was exam-ined including the heavy metal ions, polysaccharides, plant response-signaling compounds and hyperosmotic stress. Among all these elicitors,Ag+, Cd+ and YE were most effective to induce the tanshinone accumu-lation (Kai et al., 2012a; Zhao et al., 2010). After treatment with MJ andsodium nitroprusside, the four main components of tanshinones in thehairy roots increased significantly (Liang et al., 2012). In addition to thechemical and biochemical elicitors, physical or physiological stress suchas hyperosmotic stress created with sorbitol, has been shown effectiveto promote the tanshinone accumulation (Wu and Shi, 2008). T-DNA ac-tivation of taggingmutagenesis has also been applied to attain transgenichairy root lines with high tanshinone producing capacity (Lee et al.,2008).

Most of the previous studies have focused on the stimulation of theend product tanshinones in S. miltiorrhiza cell and hairy root culturesbut paid less attention to themolecularmechanism of tanshinone accu-mulation induced by various elicitors. We have recently examinedthe regulating effects of YE (biotic elicitor), Ag+ (abiotic elicitor) andMJ (elicitor signal molecule) on the tanshinone biosynthesis pathwayin S.miltiorrhiza hairy roots based on the changes of tanshinone produc-tion and the expression profiles of all the known tanshinone biosynthe-sis related genes over time (Kai et al., 2012a). A linear correlationwas observed between the gene expression and the accumulationof tanshinones, suggesting that the accumulation of tanshinones maybe activated by simultaneous up-regulation of several tanshinonebiosynthesis genes in the hairy roots during elicitor treatment. This isin agreementwith the hypothesis that elicitor-induced cellular andmo-lecular events are required for enhancement of secondary metabolitebiosynthesis in hairy roots.

Endophytic fungi of S. miltiorrhiza

Endophytes aremicrobes living entirely within the host plants with-out disturbing the normal life of host plants. Endophytic fungi have be-come a novel and important source of bioactive natural products (Louet al., 2013; Strobel, 2003). Some endophytes can produce the similarbioactive components that are originally produced in their host plants,though the physiological process for the fungi to gain this biosynthetic

ll cultures.

Reference

Chen et al. (2000a,2000b); Chen et al. (2001); Yan et al. (2005)Zhang et al. (2004)Ge and Wu (2005a)Ge and Wu (2005b); Kai et al. (2012a)Wu et al. (2007)Wu and Shi (2008)Zhao et al. (2010)

Liang et al. (2012)Yang and Ma (2012)

lycol; SA, salicylic acid; SNP, sodium nitroprusside; YE, yeast extract or yeast elicitor.



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capacity is still not known (Zhao et al., 2011). Asmicrobial cells are usu-ally much easier and faster to grow and produce the metabolites by fer-mentation than plant cells or hairy roots, it is attractive to explore themicrobial sources for the valuable compounds (Ming et al., 2012).Since the discovery of a paclitaxel-producing fungus from Taxus plantin 1993 (Stierle et al., 1993), there has been a continued interest inthe isolation of endophytic fungi for producing plant-derived bioactivecompounds from various host plants such as Taxus chinese, Camptothecaacuminata and Ginkgo biloba (Cui et al., 2012; Guo et al., 2006; Kusariet al., 2009). Only a few recent studies have been done on isolation ofendophytic fungi from S. miltiorrhiza and production of secondarymetabolites as follows.

A total of 50 species of endophytic fungi have been isolated fromS. miltiorrhiza by Wei et al. (2010), and antibacterial activity has beendetected for the metabolites of the endophytic fungi. Two of the fun-gal species, DRl2 and DR21, were found to accumulate a low contentof tanshinone II A (Wei et al., 2010). In another study (Ming et al.,2012), 18 endophytic fungal species have been isolated fromS. miltiorrhiza roots, of which one fungal species, identified as aT. atroviride fungus, accumulated tanshinone I and tanshinone IIAas detected by high-performance liquid chromatography (HPLC) andliquid chromatography-high resolution mass spectrometer/mass spec-trometry (LC-HRMS/MS). By a similar strategy, a total of 57 endophyticfungal isolates were obtained from the S. miltiorrhiza roots, some ofwhich showed strong antibacterial and antifungal activities, thoughtheir capability of tanshinone production was not reported (Lou et al.,2013). However, the contents of tanshinones found in the endothyticfungi were very low and not acceptable for industrial production. Theapplication of strain development techniques such as mutation breed-ing and optimization of the fermentation conditions may be useful toenhance the fungal production of tanshinones.

Production of tanshinones in tissue cultures

Tanshinones and other active compounds of Danshen aremainly ob-tained by extraction from dried roots of S.miltiorrhiza plants. However,the production from wild or field-grown plants is strongly dependentby environmental, ecological and climatic conditions. Plant in vitroculture system offer an alternative process for the biotechnological pro-duction of useful natural products under desired and well controlledconditions (Georgiev et al., 2013). Various tissue culture systems includ-ing callus, cell and root cultures of S. miltiorrhiza have been studied forproduction of the useful secondary metabolites in the last few decadesand summarized recently (Table 1; Dreger et al., 2010; Wang and Wu,2010). Transformed plant hairy roots offer the advantages of fast andhormone-free growth, and genetic stability, while they can producesimilar or even high level of secondary metabolites originally synthe-sized in the normal roots (Georgiev et al., 2013; Mishra and Ranjan,2008; Srivastava and Srivastava, 2007). Since the first report onS.miltiorrhiza hairy root cultures (Hu and Alfermann, 1993),many stud-ies have been documented on strain comparison,medium optimization,elite line selection and elicitation to enhance production of secondarymetabolites (Dreger et al., 2010; Wang and Wu, 2010).

The key step for successful commercial production by hairy root-based biotechnology is the large-scale cultivation in optimal bioreactors(Georgiev and Weber, 2014), though most previous studies ontanshinone production by hairy roots have been carried out in shake-flasks (Wang andWu, 2010). Large-scale production has to be achievedin bioreactors, which allows for controlled conditions to minimizevariations in the yield and product quality, and the optimization of con-ditions for efficient cell growth and secondary metabolite production(Stiles and Liu, 2013). Up to date, only a small number of valuableplant natural products have been produced in bioreactors on a commer-cial scale such as paclitaxel, shikonin, ginsenosides and berberine(Baque et al., 2012). Nevertheless, various bioreactor configurationshave been evaluated for large-scale plant cell cultures, including


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mechanically driven reactors (e.g., stirred tank, wave and rotatingdrum reactors), pneumatically driven reactors (e.g., bubble columnand airlift reactors) and bed reactors (e.g., trickle bed andmist reactors)(Georgiev et al., 2013; Weathers et al., 2010).

Since the hairy root cultures are more sensitive to shear stress, bio-reactor systems suitable for the cultivation of hairy root are differentfrom those of suspension plant-cell cultures (Mishra and Ranjan,2008). A variety of reactor configurations have been used to cultivatehairy roots, including stirred-tank bioreactors, airlift bioreactors,bubble-column bioreactors, trickle-bed bioreactors and nutrient-mistbioreactors (Mishra and Ranjan, 2008; Srivastava and Srivastava,2007; Weathers et al., 2010). The bioreactors can be divided into threetypes, liquid-phase, gas-phase, and combination of liquid and gas-phase. Qiu et al. (2004) examined the mass culture of S. miltiorrhizahairy root in a 10 L ball-shaped airlift bioreactor, achieving a 20% highergrowth index but a 30% lower tanshinone content than those in a 1-Lflask over 50 days. Similar levels of secondary metabolites wereattained in a 70 L airlift bioreactor. More recently, our group has testeda 30 L stirred tank bioreactor for S. miltiorrhiza hairy root culture, butfound difficult to maintain effective mixing and mass transfer for thehairy root growth (unpublished data). Modification and optimizationof the existing bioreactor systems and the further development ofnew bioreactor systems are needed for the cultivation of S. miltiorrhizahairy roots on a commercial scale.

ED PConclusions and future prospects

Medicinal plants are among the most attractive sources of bioactivenatural products for the development and discovery of new drugs.S. miltiorrhiza root or Danshen is an important herb not only in tradi-tional Chinese medicine but also in modern medicine for treatment ofcardiovascular diseases. As the chief bioactive constituents of Danshen,tanshinones have shown several notable pharmacological activitiesand are promising candidates for new drugs. The increasing demandfor herbal medicines and the decreasing agriculture land for medicinalplants have motivated the research effort to develop alternativemeans for production of the herbal constituents. Biotechnology meansor approaches have been among the most promising and fruitfulalternatives in this endeavor with significant progress over the last15–20 years in gene cloning in the tanshinone biosynthetic pathways,metabolic engineering, synthetic biology, and themeasures for efficientproduction in bioreactors such as hairy root culture, elicitation treat-ment and endophytic fungi.

The lack of whole genome information for S. miltiorrhiza has ham-pered the genetic engineering of tanshinone biosynthesis for a longtime. Recent advances in next-generation sequencing technology havegreatly facilitated the acquisition of transcriptome data from medicinalplants, which will be very useful for identifying the biosynthetic genesfor tanshinones in S. miltiorrhiza. The T-DNA tagging insertion of mu-tants which has become a powerful tool for gene function identificationin model plants such as Arabidopsis may be used to construct aninsertion-mutant library for S. miltiorrhiza. For the development andapplication of metabolic engineering and biotechnology approaches,hairy root culture will continue playing an important role. The variousmultiphase bioreactors developed for plant tissue and organ culturescan be applied to large scale production in hairy root cultures. The redi-rection of biosynthesis compartments, utilization of metabolomics,transcriptomics and genomics can open new opportunities and break-throughs for the application of metabolic engineering and biotechno-logical approaches in S. miltiorrhiza.

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Acknowledgments

This work was supported by National Natural Science Fund(31270007, 31201261, 30900110), New Century Talent Project (NECT-13-0902), Fok Ying-Tong Education Foundation (131041), ShanghaiScience and Technology Committee Project (10JC1412000), ShanghaiEducation Committee Fund (13ZZ104, 09ZZ138, J50401), ShanghaiTalent Development Fund, and research grants from The Hong KongPolytechnic University.

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