WITHDRAWN: Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza

JBA-06845; No of Pages 12 Biotechnology Advances xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

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Research review paper

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

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

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Keywords: Salvia miltiorrhiza Tanshinones Pharmacological activity Biosynthetic pathway Gene cloning Metabolic engineering Hairy root culture Elicitation

Laboratory of Plant Biotechnology, Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China Department of General Surgery, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai 200032, China 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 b

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Radix Salvia miltiorrhiza Bunge, generally called Danshen, is an important Chinese herb which is most effective for treatment of cardiovascular diseases and inflammations. Tanshinones, a group of abietane diterpenes, represent a major class of pharmacologically active constituents of Danshen with significant antioxidant, anti-inflammatory and anti-cancer activities, some of which have been explored as new drug candidates. Biotechnology approaches have been taken to improve the conventional processes and to develop new processes for efficient production of tanshinones so as to meet the increasing demand. The development of effective biotechnology means requires sufficient understanding of the biosynthetic pathways of tanshinones and the molecular regulation mechanisms. 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. Transformed hairy root culture of S. miltiorrhiza has been one of the most useful experimental systems for metabolic engineering studies and also a potential system for biotechnology production of tanshinones. This review summarizes the chief pharmacological activities and the recent advances in tanshinone biosynthesis and metabolic 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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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacological activities of tanshinones . . . . . . . . . . . . . . . . . Cardiovascular protection . . . . . . . . . . . . . . . . . . . . . . Antioxidant activities . . . . . . . . . . . . . . . . . . . . . . . . Anti-inflammatory activity. . . . . . . . . . . . . . . . . . . . . . Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . Cytotoxic activities and anticancer potential . . . . . . . . . . . . . . Other pharmacological activities . . . . . . . . . . . . . . . . . . . Biosynthesis and regulation of tanshinones in S. miltiorrhiza . . . . . . . . Biosynthesis of tanshinones . . . . . . . . . . . . . . . . . . . . . Cloning and characterization of genes related to tanshinone biosynthesis. Genes in the early stage . . . . . . . . . . . . . . . . . . . Genes in the middle stage. . . . . . . . . . . . . . . . . . . Genes in the late stage . . . . . . . . . . . . . . . . . . . . Regulatory mechanisms involved in tanshinone biosynthesis . . . . . . Biotechnological approaches to enhancing tanshinone production . . . . . . Metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . Elicitation treatment . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Tel./fax: +86 21 6432 1291. ⁎⁎ Corresponding author. Tel.: +852 3400 8671; fax: +852 2364 9932. E-mail addresses: [email protected] (G. Kai), [email protected] (J.-Y. Wu).

http://dx.doi.org/10.1016/j.biotechadv.2014.10.001 0734-9750/© 2014 Published by Elsevier Inc.

Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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Endophytic fungi of S. miltiorrhiza. . . . . Production of tanshinones in tissue cultures Conclusions and future prospects . . . . . . . Uncited reference . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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Salvia miltiorrhiza Bunge (family Labiatae) (Fig. 1) is an important Chinese herbal plant and its dried root, generally named Danshen in Chinese or Chinese Red Sage, has been widely used in modern and traditional 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 other herbs in China and some other countries such as Australia, Germany, and the United States (Cheng, 2007). Raw Danshen roots and formulated Danshen therapeutic products in various dosage forms have been sold in large quantities in China, among which the Fufang Danshen Dripping Pill and Fufang Danshen Tablet are the most widely distributed (Zhou et al., 2005). The Fufang Danshen Dripping Pill which is listed in the official China pharmacopeia has been widely used in China, and also in several other countries such as Russia, Cuba, the Korean Republic, Saudi Arabia and Vietnam (Zhang et al., 2012; Zhou et al., 2005). It is also the first TCM drug approved for clinical trials in the USA by the Food and Drug Administration (FDA), passed phase II trials in 2010, and approved for phase III clinical trials in 2012 on patients with chronic stable angina pectoris (http://clinicaltrials.gov/, No. NCT00797953). The market 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 of Danshen, tanshinones are a group of more than 40 abietane diterpenes such as tanshinone I, tanshinone IIA, tanshinone VI, dihydrotanshinone, and cryptotanshinone (Fig. 2) (Dong et al., 2011). Some of these tanshinones have exhibited various pharmacological activities, such as anti-oxidative, anti-inflammatory, anti-proliferative, antibacterial and anti-tumor properties (Gao et al., 2012; Gong et al., 2011; Park et al.,

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2009; Xu et al., 2010; Zhang et al., 2012), and have great potential for clinical application. Tanshinone IIA is one of the most active species and its derivative, sodium tanshinone IIA sulfate (STS), has been widely evaluated for treatment of cardiovascular diseases (CVD), cancer, hepatic fibrosis, and neurodegenerative diseases (Cheng, 2007; Takahashi et al, 2002; Wei et al., 2014). The proven therapeutic effects of Danshen and the rising public interest in herbal medicine have driven the increasing demand for the Danshen herb. Because of the scarce and extinct natural species, the commercial supply of Danshen for herbal medicine and preparation of various extracts has been mainly relied on field or domestic cultivation of the S. miltiorrhiza plants. Danshen roots on the market have been mainly produced on the agriculture farms in several Chinese provinces such as Shandong, Henan, Shanxi, Anhui, Sichuan and Hebei. The annual consumption of Danshen has exceeded 20 million kilograms (at US$2–3 per kg) in China (www. gshsyy.com). However, field cultivation of herbal plants has several disadvantages including the slow plant growth and long production period, high labor cost, occupation of farm land, and contamination by fertilizers and pesticides (Hao et al., 2014; Kai et al., 2011a). Because of the low contents of tanshinones in the field-cultivated plant roots, large amount of roots is required for the extraction and isolation of tanshinones for treatment uses (Kai et al., 2010; Liao et al., 2009). Therefore, it is of significance to develop new and more effective alternatives to the conventional processes for tanshinone production. Two of the most widely explored processes for efficient production of tanshinones and many other bioactive phytochemicals include chemical synthesis and enhanced biosynthesis through biotechnology approaches (Dong et al., 2011; Dreger et al., 2010; Kai et al., 2011a; Wang et al., 2010). Although various strategies have been developed for total organic synthesis of tanshinones (Chang et al., 1990; Danheiser et al., 1995; Wang et al., 2004), the synthetic routes are rather long, involving numerous reaction steps with very low final yields.

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Fig. 1. Live Salvia miltiorrhiza flowers (left) and roots (right) after plantation in an experimental field for two years.

Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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Antioxidant activities

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PF2401-SF, a partially purified extract of Danshen, and its main components, tanshinone I, tanshinone IIA and cryptotanshinone, can protect against liver toxicity in vivo and in vitro due to its antioxidant potential (Park et al., 2009). They can also protect against acute and sub-acute liver damage induced by carbon tetrachloride, and protect primary cultured rat hepatocytes from tertiary-butyl hydroperoxide (tBH) or D-galactosamine (GalN). In addition, tanshinone I (10 and 40 μM), tanshinone IIA (40 μM), and cryptotanshinone (40 μM) could inhibit lactate dehydrogenase leakage, glutathione (GSH) depletion, lipid peroxidation and free radical generation in vitro. PF2401-SF fortified with tanshinone I, tanshinone IIA, and cryptotanshinone showed protective effects on rat liver at a lower dose than the ethanol extract of S. miltiorrhiza due probably to its antioxidant effects (Park et al., 2009).

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Anti-inflammatory activity

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tanshinone IIA has been most widely studied and shown to prevent atherogenesis, 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 the myocardial mitochondrial membrane (Zhou et al., 2005). Tanshinone IIA also inhibited the oxidation of low-density lipoproteins (LDL), alter monocyte adhesion to the endothelium, modulate migration and proliferation of smooth muscle cell, reduce macrophage cholesterol accumulation, and reduce proinflammatory cytokine expression and platelet aggregation (Gao et al., 2012; Liu et al., 2013). Tanshinone VI can suppress hypertrophy of cardiomyocytes in neonatal 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 IGF1-untreated cells was unaltered when the cells were incubated in the presence of tanshinone VI (0.1 to 10 μM), while IGF-1-induced increases in [3H]-leucine incorporation into the trichloroacetic acid (TCA)-insoluble fraction and phosphorylated ERK were attenuated with 10 μM tanshinone VI. It appeared that tanshinone VI did not affect protein synthesis in myocardium, but attenuated myocardial hypertrophy induced by IGF-1. These findings suggest the potential of tanshinone VI as a drug candidate for improving cardiac remodeling during the development of heart failure.

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

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Pharmacological activities of tanshinones

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

Tanshinone IIA has shown anti-inflammatory activity by inhibiting the production of pro-inflammatory mediators in murine macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS) via three different pathways (Fan et al., 2009). Particularly, tanshinone IIA inhibited LPS-induced IκBα degradation and nuclear factor κB (NF-κB) activation through suppression of the NF-κB-induced kinase–IκBα kinase (NIK–IKK) pathway, the mitogen-activated protein kinases (MAPKs) pathway such as the p38 MAPK (p38) pathway, the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, and the c-Jun Nterminal kinase (JNK) pathway (Jang et al., 2006). Tanshinone IIA also inhibited LPS-induced nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production of the RAW264.7 cells and the expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) (Fan et al., 2009). These findings suggest the potential of tanshinone IIA as a selective estrogen receptor modulator (SERM) useful for the treatment of inflammation-associated neurodegenerative and cardiovascular diseases without increasing the risk of breast cancer (Fan et al., 2009).

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

Cryptotanshinone and dihydrotanshinone I have shown antibacterial activity against a number of Gram positive bacteria including Bacillus subtilis (Lee et al., 1999). A recombination-deficient mutant strain of B. subtilis was more sensitive to the two tanshinones, exhibited reduced

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Environmental impact is another major concern with the chemical methods. Therefore, it is not commercially feasible to obtain tanshinones via chemical synthesis. Therefore, various biotechnology approaches have been explored for enhancing the biosynthesis and production of tanshinones, most of which have been carried out in or through plant hairy root cultures. Although some endophytic fungi isolated from S. miltiorrhiza have been reported to produce tanshinones, the amounts are much lower than in the host plant (Ming et al., 2012). With the rapid advancement and wide application of plant biotechnology, metabolic engineering has become a feasible alternative for improving the production of targeted metabolites. Progress in this direction has been made for production of tanshinones by manipulation of the key biosynthetic genes in the S. miltiorrhiza genome using transformed hairy roots, and large scale culture of S. miltiorrhiza hairy roots or regeneration of transgenic plants (Hao et al., 2014; Kai et al., 2011a; Shi et al., 2014). This review is to summarize the recent advances in the understanding of tanshinone biosynthetic pathways, metabolic regulations, and various biotechnology approaches for more efficient production of tanshinones, and the future prospects.

Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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

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Tanshinone I has been found to ameliorate the learning and memory impairments in mice (Kim et al., 2009). Firstly, tanshinone I increased the latency time versus a vehicle-treated control group in the passive avoidance task of the test animals (Kim et al., 2009). Western blot analysis and immunohistochemical assays showed that tanshinone I increased the levels of phosphorylated cAMP response element binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase (pERK) in the hippocampus (Kim et al., 2009). Moreover, the western blot analysis showed that tanshinone I reversed the diazepam- and MK-801-induced inhibition of ERK and CREB activation in hippocampal tissues (Kim et al., 2009). The study suggests the potential of tanshinone I as a drug candidate for cognitive deficits. Dihydrotanshinone and cryptotanshinone have been shown to inhibit the acetylcholinesterase (AChE) activity in a dose-dependent manner with 50% inhibitory concentration (IC50) values of 1.0 μM and 7.0 μM, respectively (Ren et al., 2004). Therefore, these tanshinone compounds have the potential for application as a new drug to treat Alzheimer's disease (Dong et al., 2011; Ren et al., 2004). Furthermore, the lipo-hydro partition 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 been extensively evaluated, the mechanisms of action are not well understood. Moreover, the low aqueous solubility and poor membrane permeability of tashinones need to be overcome for their therapeutic applications.

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Numerous recently studies have evaluated the potential anti-tumor effects of tanshinones, mainly by in vitro cytotoxicity assays on various cancer cells such as lung, breast, prostate, colon, liver, kidney, stomach, ovary, cervix, glioma cancer cells, and melanoma, rhabdomyosarcoma and leukemia cells (Table 1). Several possible mechanisms of action have been postulated for the cytotoxic activities of tanshinones, such as anti-proliferation, pro-apoptosis, anti-angiogenesis, induction of differentiation, and inhibition of adhesion, migration, invasion and metastasis. Tanshinones may also exert the cancer cell inhibitory activities by modulating the inflammatory and immune responses, inhibiting telomerase, interacting with the DNA minor groove and activating p53 tumor suppressor, or regulating specific pathways such as the androgen receptor (AR) or signal transducer and activator of transcription (STAT3) (Liu et al., 2013; Zhang et al., 2012). Of the three tested tanshinone compounds (CT, T-IIA and T-I), tanshione I showed the most potent inhibiting activities on prostate cancer in vitro and in mice, and Aurora A kinase was identified as a possible target of the tanshinone action (Gong et al., 2011). With the notable cytotoxic activities of tanshinones on various cancer cells, the potential anticancer

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effects can be further examined in various animal models and by clinical 254 human trials before further development and application of anti-tumor 255 agents from the tanshinones. 256

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hypersensitivity in the presence of an antioxidant such as dithiothreitol (Lee et al., 1999). However, the two tanshinones showed nonselective inhibitory action on DNA, RNA, and protein synthesis in B. subtilis (Lee et al., 1999). It has been suggested that superoxide radical formation is a major cause for the antibacterial action of cryptotanshinone and dihydrotanshinone I (Lee et al., 1999). Another study has demonstrated that cryptotanshinone showed significant antibacterial activity against twenty one Staphylococcus aureus strains (Feng et al., 2009). The antibacterial action of cryptotanshinone may be related to its action as an active oxygen radical generator through transcriptome analysis, creating an oxygen-limiting state in the bacteria.

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Tanshinone I

Lung cancer Breast cancer Prostate cancer Leukemia Colon cancer Colon cancer

H1299 MCF-7, MDA-MB-231 DU145 K562 and HL-60 Colo 205 HT29 Colo 205 BT-20 MCF-7, MDA-MB-231 J5; BEL-7402 HepG2, Hep3B PLC/PRF/5 SMMC-7721 786-O MKN45, SGC7901 COC1/DDP H146 A549 LNCaP HeLa NB4 and MR2 THP-1T-cell U251 K562 mice B16/B16BL6 Rh30 HCT116 and Caco-2 HepG2 DU145 MCF-7, MDA-MB-231

Li et al., (2013) Gong et al., (2012) Gong et al., (2011) Liu et al., (2010) Su et al., (2008a) Liu et al., 2013b Su et al., 2008b Yan et al., 2012 Lu et al., 2009 Chien et al., 2012 Dai et al., 2012 Lee et al., (2010) Yuan et al., (2004) Wei et al., (2012) Chen et al., (2012) Jiao et al., (2011) Cheng et al., 2010 Chiu et al., 2010 Won et al., (2012) Pan et al., (2010) Zhang et al., (2010) Liu et al., (2009) Wang et al., (2007) Ge et al., (2012) Chen et al., (2011) Chen et al., (2010) Wang et al., (2013) Lee et al., (2009) Chuang et al., (2011) Tsai et al., (2007)

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Tanshinone IIA

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Liver cancer

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Breast cancer

Kidney cancer Stomach cancer Ovarian cancer Lung cancer Prostate cancer Cervical cancer Leukemia

Cryptotanshinone

Dihydrotanshinone I 15,16-Dihydrotanshinone I

Glioma cancer Leukemia Melanoma Rhabdo-myosarcoma Colon cancer Liver cancer Prostate cancer Breast cancer

Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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Biosynthesis of tanshinones

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Tanshinones belong to diterpenes which are biosynthesized from two major five-carbon intermediates, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be converted to one another with the catalysis of isopentenyl diphosphate isomerase (IPI). The tanshinone biosynthesis process, which is still not fully characterized, can generally be divided into three stages, starting from the formation of a universal five-carbon precursor IPP, followed by the formation of a key 20-carbon intermediate GGPP, and ending with the formation of the target product tanshinones via the late specific pathways as shown in Fig. 3. In the early stage, there are two distinct routes responsible for the synthesis of universal IPP in higher plants, the well-known mevalonate

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(MVA) pathway in the cytoplasm and the more recently discovered non-MVA 1-deoxyxylulose 5-phosphate (DXP) pathway (also called 2C-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 the MVA pathway including acetyl-CoA C-acetyltransferase (AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MDC) and, seven enzymes are involved in the DXP pathway including 1-deoxy-D xylulose5-phosphate synthase (DXS), 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR), MEP cytidylyltransferase (MCT), 4-(cytidine5diphospho)-2-C-methylerythritol kinase (CMK), 2-C-methylerythritol 2,4-cyclodiphosphate synthase (MECPS), hydroxymethybutenyl 4diphosphate synthase (HDS) and hydroxymethylbutenyl 4-diphosphate reductase (HDR) (Liao et al., 2009; Shi et al., 2014; Wang and Wu,

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Fig. 3. The biosynthetic pathways of tanshinones in S. miltiorrhiza. The solid arrows indicate known steps and the dashed arrows represent unknown steps. Enzymes in the biosynthetic pathways: AACT, acetyl-CoA C-acetyltransferase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; MK, mevalonate kinase; PMK, 5phosphomevalonate kinase; MDC, mevalonate 5-diphosphate decarboxylase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphatereductoisomerase; MCT, 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase; CMK, 4-(cytidine 5-diphospho)-2-C-methyl-Derythritolkinase; MECPS, 2-C-methylerythritol 2,4-cyclodiphosphatesynthase; HDS, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase; HDR, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase; IPPI, isopentenyl-diphosphate delta-isomerase; GGPPS, geranylgeranyl diphosphate synthase; CPS, copalyl diphosphate synthase; KSL, kaurene synthase-like; CYP76AH1, cytochrome P450 enzyme (CYP) 76AH1.

Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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Genes in the early stage AACT is the first enzyme in the MVA biosynthesis pathway, catalyzing two molecules of acetyl-CoA to acetoacetyl-CoA (Igual et al., 1992). One SmAACT cDNA (EF635969), which is comprised of a 1200-bp open reading frame (ORF) encoding a 399 amino acid protein, was cloned from S. miltiorrhiza hairy roots using a cDNA microarray and rapid amplification of cDNA ends (RACE) strategy (Cui et al., 2010). SmAACT expressed in roots, stems and leaves with higher level in the former than the other two tissues and its expression was up-regulated by both yeast extract (YE) and Ag+ elicitors as detected by cDNA microarray and quantitative RT-PCR (reverse transcription-polymerase chain reaction) techniques (Cui et al., 2010). HMGS catalyzes the condensation of acetyl-CoA with acetoacetylCoA 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-bp ORF encoding a 460 amino acid protein (Zhang et al., 2011). Expression profile analysis showed that SmHMGS was constitutively expressed in leaves, stems and roots, and was also up-regulated in response to exogenous plant hormone including salicylic acid (SA) and methyl jasmonate (MJ) (Zhang et al., 2011). HMGR conversion of 3-hydroxy-methylglutaryl-CoA (HMG-CoA) to MVA has been identified as the first key step in the MVA pathway in plants (Bach et al., 1995; Jiang et al., 2006). A full-length cDNA of SmHMGR (EU680958 in GenBank), a rate-limiting enzyme of the MVA pathway, 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 constitutively expressed gene with different levels in various tissues such as roots (high), stems (moderate) and leaves (weak). The expression of SmHMGR was up-regulated in response to exogenous SA and MJ (Liao

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Genes in the middle stage IPPI is an important enzyme in the terpenoid biosynthetic pathway (Cui et al., 2011). An IPPI gene SmIPPI with high homology with other plant IPIs has been screened out by analyzing transcriptome sequences of S. miltiorrhiza (Yang et al., 2011). The putative SmIPPI consisted of 1243 nucleotides, including a 681-bp ORF and encoding a 226 amino acid protein. It has been found by qRT-PCR analysis that SmIPPI was expressed in different developmental stages and organs, and could be induced by MJ and a fungal elicitor (Yang et al., 2011). With microarray some genes involved in tanshinone biosynthesis have been isolated and characterized, including isopentenyl diphosphate isomerase 2 (SmIPPI2) and farnesyl diphosphate synthase (SmFPS) (Cui et al., 2011). A full-length cDNA of SmGGPPS with 1234 bp has been isolated from S. miltiorrhiza by RACE (FJ643617 in GenBank), which comprised a 1092-bp ORF and encoded a 364 amino acid protein (Kai et al., 2010). SmGGPPS accelerated the biosynthesis of carotenoid in genetically engineered E. coli, suggesting that SmGGPPS was a functional protein.

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

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et al., 2009). Another full-length cDNA of SmHMGR2 (FJ747636 in GenBank) has been cloned by RACE with a 1653-bp ORF (Dai et al., 2011). SmHMGR2 was strongly expressed in leaf, stem and root tissues and could functionally complement the yeast HMGR mutant JRY2394. DXS catalyzes pyruvate and glyceraldehyde 3-phosphate to form 1deoxy-D-xylulose 5-phosphate, which is known as the first step in the DXP pathway in plants (Estévez et al., 2000; Floß et al., 2008). Two full-length cDNAs encoding 1-deoxy-D-xylulose 5-phosphate synthase (SmDXS1 and SmDXS2) were first cloned from S. miltiorrhiza by RACE in our team. The full-length cDNA of SmDXS1 (EU670744.1 in GenBank) was 2519 bp containing a 2145-bp ORF encoding 714 amino acids and the full-length cDNA of SmDXS2 (FJ643618.1 in GenBank) was 2522 bp containing a 2175-bp ORF encoding 724 amino acids. SmDXS1 was expressed in all tested tissues including roots, stems, and leaves, whereas SmDXS2 was expressed only in roots, suggesting that it is mainly involved in tanshinone biosynthesis (Kai et al., unpublished data), which is consistent with the results reported recently by Yang et al. (2013). Some other DXS members have also been found using genome-wide identification methods (Ma et al., 2012). DXR, an enzyme responsible for conversion of DXP to MEP, the second step of the DXP pathway, may play an important role in regulating the DXP pathway (Carretero-Paulet et al., 2002; Lois et al., 2000). The SmDXR gene (FJ476255 in GenBank) has been isolated and characterized in S. miltiorrhiza (Yan et al., 2009). Expression profile analysis showed that SmDXR was constitutively expressed in leaves, stems and roots, 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 function with the complementation of an Escherichia coli dxr mutant. CMK is a middle enzyme in the DXP pathway, and is the only kinase of the DXP pathway. A CMK gene (EF534309 in GenBank) was isolated from hairy roots of S. miltiorrhiza and had a 1493-bp cDNA including an 1191-bp ORF encoding a protein of 396 amino acids. The pI of deduced protein was 6.78 and its calculated molecular weight was about 43 kDa, which was similar to other reported plant CMKs (Wang et al., 2008). The expression of SmCMK and accumulation of tanshinones were both increased by MJ stimulation (Wang et al., 2008). HDR is a terminal enzyme in the DXP pathway (Hsieh et al., 2005). A full-length cDNA of SmHDR (JX233817 in GenBank) has been isolated from S. miltiorrhiza hairy roots, which consists of 1647 nucleotides with a 1392-bp ORF encoding a 463 amino acid protein (Cheng et al., 2013; Hsieh et al., 2005). The SmHDR expression appeared to be positively correlated to tanshinone accumulation in S. miltiorrhiza when treated by Ag+ (Cheng et al., 2013). Hao et al. (2013) have isolated another full-length cDNA of SmHDR from S. miltiorrhiza (JX516088 in GenBank) and identified its function in E. coli. Transcription analysis revealed that expression of SmHDR1 was high in leaves and low in roots and stems. In hairy root cultures of S. miltiorrhiza, SmHDR1 was responsive to MJ and SA, but not abscisic acid (ABA) (Hao et al., 2013).

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2010; Xu et al., 2010; Yan et al., 2009; Zhang et al., 2011). Although the MVA and DXP pathways exist in different compartments, the two pathways are interrelated and can cross talk to provide basic precursors for tanshinone biosynthesis (Ge and Wu, 2005a; Kai et al., 2011; Shi et al., 2014). In the middle stage, the key 20-carbon intermediate GGPP is derived from the universal five-carbon precursor catalyzed by geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS). GPPS catalyzes the reaction of IPP and DMAPP to form 10-carbon geranyl pyrophosphate (GPP), after which FPPS catalyzes GPP and IPP to form 15-carbon farnesyl diphosphate (FPP), and then 20-carbon GGPP is synthesized from one molecule 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 the catalysis of terpene synthases/cylases (TPSs), such as copalyl diphosphate synthase (CPS), kaurene synthase (KS), miltiradiene oxidase CYP76AH1 and other modifying enzymes (Ma et al., 2012). GGPP can be cyclized to form normal copalyl diphosphate (CPP) by copalyl diphosphate synthase (CPS), and the normal CPP catalyzed into an abietane-type diterpenoid named miltiradiene by kaurene synthaselike (KSL) through further cyclization and rearrangement (Gao et al., 2009; Xu et al., 2010). Subsequently, miltiradiene is converted to ferruginol by miltiradiene oxidase CYP76AH1 (Guo et al., 2013), and then to the target tanshinone products by others enzymes.

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Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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Although significant progress has been made in gene isolation, there is limited information about the key gene targets, the catalytic enzymes involved in the late stage, and the underlying molecular regulation mechanisms in the tanshinone biosynthetic pathway of S. miltiorrhiza. Knowledge of the key gene targets is essential for development of metabolic engineering strategies for improving the yield of tanshinones. Recently, we have explored the strategy of using differential expression profile combined with elicitation in S. miltiorrhiza hairy roots to screen the key genes in tanshinone biosynthesis (Kai et al., 2012a), and applied these 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 have been found responsive (at the transcription level) to elicitors such as MJ and YE, concurring with their induction effects on tanshinone production as reviewed below. Based on the literature data, it may be deduced that the tanshinone biosynthesis pathway is globally upregulated by certain transcription factors. The cloning of the tanshinone biosynthetic genes enables the isolation and analysis of the upstream regulation sequence, which will be helpful to understand the regulation of the gene expression and the molecular induction mechanisms for further improvement of tanshinone production. However, to our best knowledge, there are no published studies on transcription factors involved in tanshinone biosynthesis. Therefore, isolation and functional analyses of global regulatory factors on tanshinone biosynthesis as well as the regulatory mechanisms remain to be further investigated. Application of the most recent transcriptome sequencing technologies to S. miltiorrhiza (Hua et al., 2011; Yang et al., 2013; Kai et al., unpublished data) may greatly facilitate the isolation and characterization of related genes and transcription factors. Isolation and screening of the genes related to tanshinone biosynthesis is essential for development and application of biotechnological approaches for the enhancement of tanshinone production.

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Metabolic engineering

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Danshen as well as most other herbs used in traditional Chinese medicine is mainly produced by field cultivation of the medicinal plants. Field cultivation of medicinal plants is a time-consuming and laborintensive process, from which the yield and property of herb crop is often affected by environmental factors such as soil, climate, pathogens and pests. Field cultivated herb crops are also prone to variation in quality and content of active ingredients and to contamination of fertilizers, pesticides and heavy metals. Application of plant biotechnology is among the most attractive and effective measures for overcoming these problems and achieving efficient and sustainable production of medicinal plants and their bioactive constituents. This part is on the biotechnological approaches for enhanced production of tanshinones with the cell and hairy root cultures and endophytic fungi derived from S. miltiorrhiza.

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Genes in the late stage A 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 has been deduced to be composed of 793 amino acids with a pI of 6.45 and a molecular weight of 90.36 kDa. After MJ elicitation, the expression of SmCPS was strongly stimulated as well as the contents of tanshinone IIA and cryptotanshinone. The kaurene synthase-like (KSL) gene located downstream of SmCPS has 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 ORF was obtained, which encoded a 595 amino acid protein, and the molecular weight and pI were estimated to be 68.3 kDa and 6.0, respectively (Gao et al., 2009). A positive correlation was observed between SmKSL expression and the accumulation of tanshinone IIA and cryptotanshinone in S. miltiorrhiza hairy roots subjected to MJ treatment (Gao et al., 2009). Recently, by a stable isotope labeling method and next-generation sequencing approach, six candidate Cytochrome P450 enzymes (CYPs) genes have been found to be co-regulated with the diterpene synthase genes in both the rhizomes and hairy roots of S. miltiorrhiza (Guo et al, 2013). CYP76AH1, one of six CYP genes, has been successfully demonstrated to catalyze a unique oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo (Guo et al., 2013). Based on previous miltiradiene-producing Saccharomyces cerevisiae, introduction of CYP76AH1 led to heterologous production of ferruginol at 10.5 mg/L in yeast. This is of significance for further elucidating the biosynthesis of tanshinones and for the production of terpenoids in the constructed microbial cell factories in the future.

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With the successful isolation of several genes involved in the tanshinone biosynthesis pathways in recent years, it is more feasible to apply metabolic engineering for improving tanshinone production in the transformed hairy roots (Kai et al., 2011a; Shi et al., 2014). Through a push–pull strategy, the production of tanshinones has been significantly enhanced by up to 4.74-fold in a transgenic line HG9 coexpressing of SmHMGR and SmGGPPS in comparison with the control and a single-gene over-expression hairy root line (Kai et al., 2011a). Meanwhile, co-expression of SmHMGR and SmDXR has also increased tanshinone production in S. miltiorrhiza hairy root lines, and further improved after elicitation by yeast extract and/or Ag+ as reported recently (Shi et al., 2014). The hairy root culture derived from infection of the plant tissues by Agrobacterium rhizogenes bacterium carrying the Ri T-DNA plasmid has been one of the most useful systems for introducing foreign genes into the host plant. Hairy root cultures have been widely used as potential production systems for plant secondary metabolites and also convenient experimental systems for studying the metabolic processes and physiological responses to pathogens, stress and elicitors under well-controlled conditions (Li et al., 2008; Georgiev et al., 2012; Kai et al., 2012b; Hao et al., 2014). Compared to the extraction from natural plant sources and the production of secondary metabolites in plant tissue cultures, it may be more efficient to produce the desired metabolites by genetically engineered microbial cells. Based on analysis of substrate availability, metabolic flux and the energetics of resource utilization for isoprenoid biosynthesis in genetically engineered yeast S. cerevisiae with endogenous deregulated erg9 and/or coq1 gene, Huang et al. (2013) found that isoprenoid biosynthesis was closely related to mitochondrial function. With the construction of a miltiradiene synthetic pathway in S. cerevisiae using a pathway engineering strategy, four consecutive genes including SmCPS, SmKSL, GGPPs and FPS (farnesyl diphosphate synthase) have been fused and introduced into the yeast cell (Zhou et al., 2012). The miltiradiene production was significantly enhanced in the engineered yeast cell lines, e.g. to a maximal yield of 365 mg/L in a 15 L bioreactor with the YJ2X strain, whereas byproduct accumulation was reduced (Zhou et al., 2012). This study has demonstrated the promising potential for producing the target metabolites (tanshinones) by successively stacking the specific genes involved in the late stage of the tanshinone biosynthesis pathway into genetically modified yeast. Metabolic engineering in S. miltiorrhiza plant and microbial cell factories is expected to make new breakthroughs for enhanced tanshinone production. However, it requires sufficient understanding of the tanshinone biosynthesis pathways and the genetic regulation mechanisms. Moreover, the synthetic biology strategy is a promising new and powerful tool for enhancing tanshinone production in plant or microbial cell factories, as envisaged from the successful, high-yield

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

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Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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

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Elicitation, the treatment of plant cells with biotic and abiotic elicitors, is one of the most common and effective strategies for stimulating secondary metabolite production in plant tissue cultures (Wang and Wu, 2013). Elicitor is a term originated from plant science which generally refers to the agents or stimuli that can induce phytoalexin synthesis and defense responses in the challenged host plant. Therefore, the efficacy of elicitation is on the basis that the accumulation of most secondary metabolites in plants is part of the defense responses of plants to pathogen infection and environmental stresses. A variety of biotic or abiotic elicitors have been used to enhance the tanshinone production in S. miltiorrhiza hairy root and cell cultures, such as yeast extract (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 extract aqueous solution (composed mainly of carbohydrate polymers with protein) was applied to induce and stimulate the accumulation of seconday metabolites (tanshinone and phenolic acids) in liquid cultures of S. miltiorrhiza hairy roots and transformed cells. YE has then been used as an effective elicitor in several later studies, separately or in combination with 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), having an important role in activating the defense response of plants against pathogens (Jakab et al., 2001), was able to increase the tanshinone accumulation 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). It was suggested that BABA potentiated the elicitation of S. miltiorrhiza hairy roots by YE, leading to more significant enhancement of tanshinone production than each used alone. In other studies, YE was applied in combination with the silver ion Ag+ to the S. miltiorrhiza hairy root culture leading to more significant increase of tanshinone production (Ge and Wu, 2005a; Kai et al., 2012a). Based on specific pathway inhibitor experiments, tanshinone accumulation was suggest to undergo through the DXP pathway mainly as inferred by Ag+ and YE treatment (Ge and Wu, 2005a), which was further confirmed by transgenic experiment in our group (Kai et al., 2011a). The combination of elicitor treatment with effective process strategies, YE, in situ adsorption and recovery of tanshinones with a hydrophobic polymeric resin, and periodic replenishment of fresh medium, led to more dramatic increase in the tanshinione production (Yan et al., 2005). Bacillus cereus, a plant growth-promoting rhizobacterium, was inoculated into the S. miltiorrhiza hairy root culture to form a root-bacterial co-culture system, in which the total tanshinone content of roots was

t2:1 t2:2

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

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Endophytic fungi of S. miltiorrhiza

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Endophytes are microbes living entirely within the host plants without disturbing the normal life of host plants. Endophytic fungi have become a novel and important source of bioactive natural products (Lou et al., 2013; Strobel, 2003). Some endophytes can produce the similar bioactive components that are originally produced in their host plants, though the physiological process for the fungi to gain this biosynthetic

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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 secondary metabolites in hairy root cultures. The enhanced tanshinone production in the hairy root–bacteria co-culture system was attributed to the elicitor compounds such as polysaccharides released from the bacteria (Wu et al., 2007). The polysaccharide fraction of an endophytic fungus Trichoderma atroviride isolated from the root of S. miltiorrhiza has also been shown to be an effective elicitor for stimulating the tanshinone accumulation and the growth of S. miltiorrhiza hairy roots as well as the transcription of genes in the tanshinone biosynthesis pathway (Ming et al., 2013). In S. miltiorrhiza cell culture the effect of several elicitors was examined including the heavy metal ions, polysaccharides, plant responsesignaling compounds and hyperosmotic stress. Among all these elicitors, Ag+, Cd+ and YE were most effective to induce the tanshinone accumulation (Kai et al., 2012a; Zhao et al., 2010). After treatment with MJ and sodium nitroprusside, the four main components of tanshinones in the hairy roots increased significantly (Liang et al., 2012). In addition to the chemical and biochemical elicitors, physical or physiological stress such as hyperosmotic stress created with sorbitol, has been shown effective to promote the tanshinone accumulation (Wu and Shi, 2008). T-DNA activation of tagging mutagenesis has also been applied to attain transgenic hairy root lines with high tanshinone producing capacity (Lee et al., 2008). Most of the previous studies have focused on the stimulation of the end product tanshinones in S. miltiorrhiza cell and hairy root cultures but paid less attention to the molecular mechanism of tanshinone accumulation induced by various elicitors. We have recently examined the regulating effects of YE (biotic elicitor), Ag+ (abiotic elicitor) and MJ (elicitor signal molecule) on the tanshinone biosynthesis pathway in S. miltiorrhiza hairy roots based on the changes of tanshinone production and the expression profiles of all the known tanshinone biosynthesis related genes over time (Kai et al., 2012a). A linear correlation was observed between the gene expression and the accumulation of tanshinones, suggesting that the accumulation of tanshinones may be activated by simultaneous up-regulation of several tanshinone biosynthesis genes in the hairy roots during elicitor treatment. This is in agreement with the hypothesis that elicitor-induced cellular and molecular events are required for enhancement of secondary metabolite biosynthesis in hairy roots.

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t2:3

Elicitora

Culture system

Reference

t2:4 Q1 t2:5 Q2 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13

YE Ag+ BABA, BABA + YE Ag+,YE Bacillus cereus bacteria Osmotic stress (sorbitol) Heavy metal ions, polysaccharides from Bacillus cereus, SA,MJ, osmotic stress (sorbitol) MJ, SNP PEG, ABA and MJ

Hairy root Hairy root Hairy root Hairy root Hairy root Hairy root Cell

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)

Hairy root Hairy root

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

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a

ABA, abscisic acid; BABA, β-aminobutyric acid; MJ, methyl jasmonate; PEG, polyethylene glycol; SA, salicylic acid; SNP, sodium nitroprusside; YE, yeast extract or yeast elicitor.

Please cite this article as: Kai G, et al, Metabolic engineering and biotechnological approaches for production of bioactive diterpene tanshinones in Salvia miltiorrhiza, Biotechnol Adv (2014), http://dx.doi.org/10.1016/j.biotechadv.2014.10.001

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Tanshinones and other active compounds of Danshen are mainly obtained by extraction from dried roots of S. miltiorrhiza plants. However, the production from wild or field-grown plants is strongly dependent by environmental, ecological and climatic conditions. Plant in vitro culture system offer an alternative process for the biotechnological production of useful natural products under desired and well controlled conditions (Georgiev et al., 2013). Various tissue culture systems including callus, cell and root cultures of S. miltiorrhiza have been studied for production of the useful secondary metabolites in the last few decades and summarized recently (Table 1; Dreger et al., 2010; Wang and Wu, 2010). Transformed plant hairy roots offer the advantages of fast and hormone-free growth, and genetic stability, while they can produce similar or even high level of secondary metabolites originally synthesized in the normal roots (Georgiev et al., 2013; Mishra and Ranjan, 2008; Srivastava and Srivastava, 2007). Since the first report on S. miltiorrhiza hairy root cultures (Hu and Alfermann, 1993), many studies have been documented on strain comparison, medium optimization, elite line selection and elicitation to enhance production of secondary metabolites (Dreger et al., 2010; Wang and Wu, 2010). The key step for successful commercial production by hairy rootbased biotechnology is the large-scale cultivation in optimal bioreactors (Georgiev and Weber, 2014), though most previous studies on tanshinone production by hairy roots have been carried out in shakeflasks (Wang and Wu, 2010). Large-scale production has to be achieved in bioreactors, which allows for controlled conditions to minimize variations in the yield and product quality, and the optimization of conditions for efficient cell growth and secondary metabolite production (Stiles and Liu, 2013). Up to date, only a small number of valuable plant natural products have been produced in bioreactors on a commercial scale such as paclitaxel, shikonin, ginsenosides and berberine (Baque et al., 2012). Nevertheless, various bioreactor configurations have been evaluated for large-scale plant cell cultures, including

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mechanically driven reactors (e.g., stirred tank, wave and rotating drum reactors), pneumatically driven reactors (e.g., bubble column and airlift reactors) and bed reactors (e.g., trickle bed and mist reactors) (Georgiev et al., 2013; Weathers et al., 2010). Since the hairy root cultures are more sensitive to shear stress, bioreactor systems suitable for the cultivation of hairy root are different from those of suspension plant-cell cultures (Mishra and Ranjan, 2008). A variety of reactor configurations have been used to cultivate hairy roots, including stirred-tank bioreactors, airlift bioreactors, bubble-column bioreactors, trickle-bed bioreactors and nutrient-mist bioreactors (Mishra and Ranjan, 2008; Srivastava and Srivastava, 2007; Weathers et al., 2010). The bioreactors can be divided into three types, liquid-phase, gas-phase, and combination of liquid and gasphase. Qiu et al. (2004) examined the mass culture of S. miltiorrhiza hairy root in a 10 L ball-shaped airlift bioreactor, achieving a 20% higher growth index but a 30% lower tanshinone content than those in a 1-L flask over 50 days. Similar levels of secondary metabolites were attained in a 70 L airlift bioreactor. More recently, our group has tested a 30 L stirred tank bioreactor for S. miltiorrhiza hairy root culture, but found difficult to maintain effective mixing and mass transfer for the hairy root growth (unpublished data). Modification and optimization of the existing bioreactor systems and the further development of new bioreactor systems are needed for the cultivation of S. miltiorrhiza hairy roots on a commercial scale.

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capacity is still not known (Zhao et al., 2011). As microbial cells are usually much easier and faster to grow and produce the metabolites by fermentation than plant cells or hairy roots, it is attractive to explore the microbial sources for the valuable compounds (Ming et al., 2012). Since the discovery of a paclitaxel-producing fungus from Taxus plant in 1993 (Stierle et al., 1993), there has been a continued interest in the isolation of endophytic fungi for producing plant-derived bioactive compounds from various host plants such as Taxus chinese, Camptotheca acuminata and Ginkgo biloba (Cui et al., 2012; Guo et al., 2006; Kusari et al., 2009). Only a few recent studies have been done on isolation of endophytic fungi from S. miltiorrhiza and production of secondary metabolites as follows. A total of 50 species of endophytic fungi have been isolated from S. miltiorrhiza by Wei et al. (2010), and antibacterial activity has been detected for the metabolites of the endophytic fungi. Two of the fungal species, DRl2 and DR21, were found to accumulate a low content of tanshinone II A (Wei et al., 2010). In another study (Ming et al., 2012), 18 endophytic fungal species have been isolated from S. miltiorrhiza roots, of which one fungal species, identified as a T. atroviride fungus, accumulated tanshinone I and tanshinone IIA as detected by high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometer/mass spectrometry (LC-HRMS/MS). By a similar strategy, a total of 57 endophytic fungal isolates were obtained from the S. miltiorrhiza roots, some of which showed strong antibacterial and antifungal activities, though their capability of tanshinone production was not reported (Lou et al., 2013). However, the contents of tanshinones found in the endothytic fungi were very low and not acceptable for industrial production. The application of strain development techniques such as mutation breeding and optimization of the fermentation conditions may be useful to enhance the fungal production of tanshinones.

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Medicinal plants are among the most attractive sources of bioactive natural products for the development and discovery of new drugs. S. miltiorrhiza root or Danshen is an important herb not only in traditional Chinese medicine but also in modern medicine for treatment of cardiovascular diseases. As the chief bioactive constituents of Danshen, tanshinones have shown several notable pharmacological activities and are promising candidates for new drugs. The increasing demand for herbal medicines and the decreasing agriculture land for medicinal plants have motivated the research effort to develop alternative means for production of the herbal constituents. Biotechnology means or approaches have been among the most promising and fruitful alternatives in this endeavor with significant progress over the last 15–20 years in gene cloning in the tanshinone biosynthetic pathways, metabolic engineering, synthetic biology, and the measures for efficient production in bioreactors such as hairy root culture, elicitation treatment and endophytic fungi. The lack of whole genome information for S. miltiorrhiza has hampered the genetic engineering of tanshinone biosynthesis for a long time. Recent advances in next-generation sequencing technology have greatly facilitated the acquisition of transcriptome data from medicinal plants, which will be very useful for identifying the biosynthetic genes for tanshinones in S. miltiorrhiza. The T-DNA tagging insertion of mutants which has become a powerful tool for gene function identification in model plants such as Arabidopsis may be used to construct an insertion-mutant library for S. miltiorrhiza. For the development and application of metabolic engineering and biotechnology approaches, hairy root culture will continue playing an important role. The various multiphase bioreactors developed for plant tissue and organ cultures can be applied to large scale production in hairy root cultures. The redirection of biosynthesis compartments, utilization of metabolomics, transcriptomics and genomics can open new opportunities and breakthroughs for the application of metabolic engineering and biotechnological approaches in S. miltiorrhiza.

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This work was supported by National Natural Science Fund (31270007, 31201261, 30900110), New Century Talent Project (NECT13-0902), Fok Ying-Tong Education Foundation (131041), Shanghai Science and Technology Committee Project (10JC1412000), Shanghai Education Committee Fund (13ZZ104, 09ZZ138, J50401), Shanghai Talent Development Fund, and research grants from The Hong Kong Polytechnic University.

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