Regulation and metabolic engineering of tanshinone biosynthesis

Abstract

Salvia miltiorrhiza (Chinese name: dānshēn) is used in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. The tanshinones represent the most important biological active class of compounds present in dānshēn extracts. They are synthesized via either the cytoplasmic mevalonate or the plastidial 2-C-methyl-D-erythritol-4-phosphate pathway. Here, we summarize recent discoveries regarding the mechanisms underlying tanshinone biosynthesis and how the process is regulated. Tanshinone accumulation in planta is affected by a range of elicitors and by the composition of the culture medium. Its production in hairy root cultures can be enhanced by the over-expression of genes encoding 1-deoxy-D-xylulose 5-phosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranyl diphosphate synthase and allene oxide cyclase. The pathway leading to the biosynthesis of the tanshinone precursors miltiradiene and ferruginol, has been engineered in yeast.

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1. Introduction

Salvia species have been used in herbal medicine in many parts of the world, thanks to their content of a range of medicinally active compounds. The root of S. miltiorrhiza (dānshēn in Chinese), for example, is a highly prized ingredient of a number of Asian herbal medicine preparations; it is associated with curative properties against coronary artery disease, angina, myocardial infarction, cerebrovascular disease, hepatitis, chronic renal failure and dysmenorrhea.^1,2 As many as 320 dānshēn-containing preparations are manufactured by various pharmaceutical concerns.³ Some of these have lately become available outside of China, and represent the first traditional Chinese medicines to be approved for clinical trials by the US Food and Drug Administration. Its recognized medicinal value has generated a steady rise in demand for dānshēn, and some 80 kt per year are currently being produced in China for domestic use and the export trade.⁴ A number of Salvia spp. are currently exploited in herbal medicine concoctions. S. aegyptiaca is an ingredient in formulations directed against numerous complaints, including diarrhoea, gonorrhoea, haemorrhoids, eye diseases, nervous disorders, dizziness and trembling; S. bucharica against liver ailment; S. cavaleriei against dysentery, boils and superficial wounds; S. desoleria against menstrual, digestive and central nervous system diseases; S. officinalis against tuberculosis, psoriasis and seborrhoeic eczema; S. parryi against various stomach disorders; S. przewalskii against cardiovascular problems; and finally S. yunnanensis against both the acquired immune deficiency syndrome (AIDS) and hepatitis B viruses.^5–10

The two major classes of active compounds present in dānshēn are the lipid-soluble tanshinones and the water-soluble phenolic acids. The tanshinones are abietane diterpenes, and were first isolated by Nakao in 1930 from dānshēn roots, from which, in the intervening period, more than 40 other diterpenes have been identified.¹¹ Tanshinones have been detected throughout the plant, with the exception of the seed. In the root, they accumulate preferentially in the cortex, while in the above ground part of the plant, they are found largely in the epidermis.^12–14 The compounds have been shown to be associated with a range of pharmaceutical activities. The five most important groups of tanshinone, as defined by their functionality (Fig. 1), are tanshinone I (TI), tanshinone IIA (TIIA), tanshinone IIB (TIIB), cryptotanshinone (CT) and dihydrotanshinone I (DTI). TI suppresses the growth of breast cancer cells through its interaction with adhesion molecules;¹⁵ TIIA inhibits osteoclast differentiation and affects the bone resorptive activity of differentiated osteoclasts;¹⁶ TIIB exhibits neuro-protective activity in rats;¹⁷ CT inhibits the growth of oral bacteria;¹⁸ and DTI suppresses endothelial cell proliferation, migration, invasion and tube formation.¹⁹

Fig. 1 Chemical structure of tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone and dihydrotanshinone I.

Traditional methods of tanshinone production are inadequate to meet the rapidly rising demand, largely because yield levels are low and the plants are very slow-growing. Here, the prospects for biotechnological intervention aimed at increasing tanshinone production are discussed, focusing on the use of elicitors, the manipulation of culture conditions, and genetic engineering.

2. The tanshinone synthetic pathway in dānshēn roots

The tanshinone biosynthesis pathway is complex and has been only partially elucidated so far. A multi-platform genomics-based approach has recently been initiated, based on the construction of an expressed sequence tag (EST) library, the application of Solexa sequencing and 454 GS-FLX transcriptome pyrosequencing, the use of cDNA microarray technology and cDNA-AFLP profiling.^20–24 The EST library was generated from mRNA extracted from whole plantlets, and comprises 10 228 sequences;²⁰ the Solexa-based transcriptome sampled from the entire plant life cycle has defined 56 774 unigenes;²¹ the 454 GS-FLX pyrosequencing has generated a set of 64 139 unigenes present in the root and leaf;²² the cDNA microarray analysis has identified 114 differentially transcribed cDNAs in hairy root cultures;²³ and the cDNA-AFLP profiling, using 128 primer pairs, has revealed that 2300 transcript-derived fragments (TDFs) were differentially expressed among S. miltiorrhiza and S. castanea.²⁴ Inspection of a genome draft genome sequence (not currently available in the public domain) has identified 40 terpenoid biosynthesis-related genes, the products of which include enzymes involved in the biosynthesis of the precursors of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).²⁵

The tanshinones are synthesized from the central five-carbon intermediate IPP, which is itself generated via either the cytoplasmic mevalonic acid (MVA) pathway or the plastidial 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the MVA pathway. This enzyme catalyses the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. The enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is subsequently reduced to yield MVA in the presence of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). Mevalonate kinase (MK) phosphorylates MVA into mevalonate-5-phosphate (MVAP), and a further phosphorylation into mevalonate diphosphate (MVAPP) is catalysed by 5-phosphomevalonate kinase (PMK). The transformation of MVAPP to IPP involves the enzyme MVAPP decarboxylase (MDC). The initial step in the MEP pathway is the condensation of pyruvate and glyceraldehyde 3-phosphate (GA-3P) to form 1-deoxy-D-xylulose 5-phosphate (DXP), catalysed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS). In the presence of DXP reductoisomerase (DXR), DXP is reduced to MEP and subsequently is transformed through the action of the enzyme 2-C-methyl-D-erythritol-4-phosphate cytidyl transferase (MCT) into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME). Later steps in the pathway comprise the mediated by CDP-ME kinase (CMK)-mediated phosphorylation of CDP-ME into CDP-ME-2-phosphate (CDP-MEP), which provides the substrate for the action of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (MDS) to form 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (cMEPP). cMEPP in turn is converted by the enzyme 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP) synthase (HDS) into HMBPP, and finally, HMBPP reductase (HDR) catalyses the formation of IPP and dimethylallyl-diphosphate (DMAPP). The conversion of IPP to DMAPP and the equilibrium concentrations of IPP and DMAPP are controlled by the enzyme isopentenyl-diphosphate deltaisomerase (IPPI). Geranyl diphosphate synthase (GPPS) condenses IPP to DMAPP to form geranyl pyrophosphate (GPP). The condensation of IPP and GPP to form farnesyl diphosphate (FPP) is mediated by farnesyl diphosphate synthase (FPPS). Geranylgeranyl diphosphate synthase (GGPPS) condenses IPP with FPP to form geranylgeranyl diphosphate (GGPP).^26,27 Copalyl diphosphate synthase (CPS), a class II diterpene cyclase, converts GGPP to copalyl diphosphate (CPP), whereas ent-kaurene synthase-like (KSL), a class I diterpene cyclase, produces the abietane miltiradiene class of diterpenes.²⁸ Miltiradiene is transformed to ferruginol though the action of the cytochrome P450 monooxygenase CYP76AH1 (Fig. 2).²⁹

Fig. 2 A proposed pathway for tanshinone biosynthesis in dānshēn (modified from Yang et al.,²² Ma et al.,²⁵ Gao et al.²⁸ and Guo et al.²⁹). HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA; MVA: mevalonic acid; MVAP: mevalonate-5-phosphate; MVAPP: mevalonate diphosphate; GA-3P: glyceraldehyde 3-phosphate; DXP: 1-deoxy-D-xylulose 5-phosphate; MEP: 2-C-methyl-D-erythritol-4-phosphate; CDP-ME: 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol; CDP-MEP: 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol 2-phosphate; cMEPP: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBPP: 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate; IPP: isopentenyl diphosphate; DMAPP: dimethylallyl diphosphate; GPP: geranyl pyrophosphate; FPP: farnesyl diphosphate; GGPP: geranylgeranyl diphosphate; CPP: copalyl diphosphate; AACT: acetyl-CoA C-acetyltransferase; HMGS: 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR: 3-hydroxy-3-methylglutaryl-CoA reductase; MK: mevalonate kinase; PMK: 5-phosphomevalonate kinase; MDC: mevalonate 5-diphosphate decarboxylase; DXS: 1-deoxy-D-xylulose 5-phosphate synthase; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; MCT: 2-C-methyl-D-erythritol-4-phosphate cytidyl transferase; CMK: 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol kinase; MDS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; 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 deltaisomerase; GPPS: geranyl diphosphate synthase; FPPS: farnesyl diphosphate synthase; GGPPS: geranylgeranyl diphosphate synthase; CPS: copalyl diphosphate synthase; KSL: ent-kaurene synthase-like.

A set of 14 of the dānshēn genes involved in the MVA and MEP pathways, namely SmAACT1, SmHMGS, SmHMGR1, SmHGMR2, SmHGMR3, SmDXR, SmCMK, SmMDS, SmHDR1, SmGGPPS, SmFPPS, SmCPS1, SmKSL1 and SmCYP76AH1 has been isolated in recent years (Table 1).^23,30–42 Kai et al.⁴³ successfully demonstrated that the activity of SmHMGR, SmDXS2, SmFPPS, SmGGPPS and SmCPS is important for the accumulation of tanshinones in hairy root cultures of dānshēn, and have suggested that these five genes could function as rate-limiting genes in the tanshinone biosynthesis pathway. The cyclization of GGPP into diterpenoids is achieved by the action of a number of bifunctional synthases, including the miltiradiene synthases SmCPS and SmKSL. Since SmKSL co-precipitates with SmCPS in vitro, the assumption is that these two enzymes interact directly with one another in vivo, possibly through the formation of an enzyme complex. Protein modeling has demonstrated that the active sites in an SmKSL–SmCPS complex are more closely associated with one another than are those in SmCPS–SmKSL.⁴⁴

Table 1 Tanshinone biosynthesis-related genes in dānshēn

Name	Accession no.	Gene length (bp)	Protein length (aa)	Reference
SmDXS1	EU670744	2519	714	25
SmDXS2	FJ643618	2522	724	25
SmDXS3	JN831116	2142	713	25
SmDXS4	JN831117	2142	713	25
SmDXS5	JN831118	2112	703	25
SmDXR	FJ476255	1425	474	30
	DQ991431	1665	474	31
SmMCT	JN831096	915	304	25
SmCMK	EF534309	1477	396	32
SmMDS	JN831097	705	234	25
	JX233816	988	234	33
SmHDS	JN831098	2229	742	25
SmHDR1	JN831099	1392	463	25
	JX233817	1647	463	34
SmHDR2	JN831100	1389	462	25
SmAACT1	EF635969	1569	399	35
SmAACT2	JN831101	1212	403	25
SmHMGS	FJ785326	1655	460	36
SmHMGR1	EU680958	2115	565	37
SmHMGR2	FJ747636	1653	550	38
SmHMGR3	JN831102	1689	562	39
SmHMGR4	JN831103	1653	550	25
SmMK	JN831104	1164	387	25
SmPMK	JN831095	1530	509	25
SmMDC	JN831105	1269	422	25
SmIPPI1	EF635967	1234	305	23
SmIPPI2	JN831106	810	269	25
SmGPPS	JN831107	1275	424	25
SmFPPS	EF635968	1494	349	23
	HQ687768	1319	349	40
SmGGPPS1	FJ643617	1563	364	41
	FJ178784	1563	364	42
SmGGPPS2	JN831112	1041	346	25
SmGGPPS3	JN831113	1140	379	25
SmCPS1	EU003997	2613	793	28
SmCPS2	JN831114	2274	757	25
SmCPS3	JN831115	2106	701	25
SmCPS4	JN831120	1983	660	25
SmCPS5	JN831121	1338	445	25
SmKSL1	EF635966	2110	595	28
SmKSL2	JN831119	2289	762	23
SmCYP76AH1	JX422213	1488	495	29

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3. Regulation of tanshinone biosynthesis in dānshēn

3.1. Biotic elicitors

Song et al.⁴⁵ have shown that an elicitor produced by Armillaria mellea is able to induce tanshinone accumulation in a dānshēn crown gall culture. Certain oligosaccharins are effective in both promoting hairy root growth and stimulating tanshinone biosynthesis.⁴⁶ Yeast elicitor (the carbohydrate fraction of yeast extract) increased the accumulation of tanshinones in dānshēn hairy root and cell suspension cultures.^43,47–53 Yeast elicitor has a marked positive effect on the growth of hairy roots,⁴⁸ although this result could not be confirmed by Ge and Wu.⁵⁰ This discrepancy may, however, been due to differences in the amount of time allowed. A fungal elicitor derived from a konjac endophytic fungus has been observed to inhibit hairy root growth, but at the same time to enhance the biosynthesis of DTI and CT.⁵⁴ Both an extract of the mycelium and the polysaccharide fraction of the endophytic fungus Trichoderma atroviride can promote hairy root growth and stimulate the production of tanshinones.⁵⁵ Yeast extract, certain oligogalacturonides and particularly an elicitor produced by the fungus Fusarium oxysporum, can all increase tanshinone yield.⁴⁶ Wu et al.⁵⁶ have reported that the tanshinone content of hairy roots can be increased by at least 12 fold by co-cultivation with live Bacillus cereus cells, although the growth of the hairy roots was significantly inhibited. The polysaccharide fraction of Bacillus cereus stimulates tanshinone accumulation in hairy roots by about seven-fold, while the protein fraction promotes hairy root growth.⁵⁷ Streptomyces pactum Act12 has a certain promotional effect on the growth of hairy root at an appropriate concentration and increases the accumulation level of tanshinone in hairy roots.⁵⁸ A chito-oligosaccharide plant growth regulator is known to significantly enhance the accumulation of TIIA, as well as to promote plant and root growth.⁵⁹

3.2. Abiotic elicitors

Supplementation of growth medium with various metal ions can promote tanshinone biosynthesis in dānshēn hairy roots and seedlings.^{43,49,51,52,60–65} both Cu²⁺ and Zn²⁺ have a positive effect on biomass accumulation,⁶⁰ while Ag⁺ and Co²⁺ have a negative one.^51,61 Mn²⁺ has a dose-dependent impact on seedling growth.⁶² The provision of 100 μM sodium nitroprusside (SNP), which supplies a nitrogen oxide moiety, also enhances tanshinone production.⁶³ Some plant growth regulators, such as methyl jasmonate (MJ), indol-yl-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), gibberellic acid (GA3), 6-benzylaminopurine (6-BA), thidiazuron (TDZ) and abscisic acid (ABA), induced tanshinone accumulation in dānshēn hairy roots and seedlings.^{43,49,54,66–72} Gupta et al.⁶⁹ have shown that hairy root growth is stimulated by the inclusion of both TDZ and 6-BA, while according to Sun et al.,⁶⁷ GA3 promotes the growth of the aerial part of the dānshēn plant, but inhibits the growth of the root. The above-ground and underground biomass of dānshēn increased with the increasing of IAA concentration and then decreased. The impact of MJ and ABA on the growth of dānshēn is inconclusive; according to Ge and Wu,⁵⁰ MJ stimulates the growth of hairy roots, but this conclusion was not borne out in other experiments.^63,66 Gupta et al.⁶⁹ were unable to demonstrate any effect of supplying hairy roots with ABA, while Sheng and Zhu claimed that the phytohormone has a negative impact.⁷² The provision of β-aminobutyric acid (BABA), α-animo isobutyric acid (AIB) or sodium nitroprusside all increase the level of tanshinone production in hairy root cultures, as well as enhancing hairy root growth.^50,52,69 Shi et al.⁷³ and Wu and Shi⁵³ have shown that supplementing hairy root cultures with sorbitol also enhances their tanshinone content, as does that of polyethylene glycol (PEG)-6000, although in the latter case, the supplement suppresses biomass accumulation.^70,71,74

3.3. Other treatments

Moisture stress favors the accumulation of TI, TIIA, CT and DTI. Severe drought reduces the shoot and root biomass of dānshēn plants, although an episode of mild drought appears to have the opposite effect.^75,76 Tanshinone content responds negatively to an increase in nitrogen availability.^77,78 Han and Liang have shown that that the greater the quantity of available phosphorus, the higher the TIIA content of the plant.⁷⁸

3.4. Combinations of elicitors and treatments

Some biotic elicitors and abiotic elicitors have been shown to act synergistically to regulate tanshinone biosynthesis. The exposure of hairy root dānshēn cultures to Ag⁺ prior to supplementation by yeast extract amplifies the enhancing effect of the supplement.⁴⁹ When yeast extract and Ag⁺ are supplied simultaneously, the accumulation of TI is encouraged; combining yeast extract and Co²⁺ favors TIIA content, and the combination of yeast extract with AIB promotes CT accumulation.⁵² Both the provision of sucrose and the replacement of the medium prior to the addition of Ag⁺ reverses the growth inhibition, significantly increasing biomass concentration and tanshinone yield.⁶¹ Ge and Wu were able to demonstrate that combining yeast extract with BABA or MJ increases the production of tanshinones, but only when hormones are given a few days before the provision of yeast extract.⁵⁰ Yeast extract and sorbitol appear to act synergistically, since the provision of both simultaneously has a larger effect than the provision of either additive on its own.^53,72 Combinations of yeast extract and the various elicitors do not inhibit the growth of dānshēn hairy roots; in fact, the combination yeast extract plus sorbitol even promotes the expansion of the root biomass.^{43,49,50,52,53,73} Combining a fungal elicitor with MJ similarly increases tanshinone content to higher levels than either the fungal elicitor or MJ on its own.⁵⁴ A combination of low level IAA and GA also promoted increase in both dānshēn biomass and tanshinone content.⁶⁷ The total content of tanshinone IIA in hairy roots is stimulated by the presence of 0.2 mg L⁻¹ NAA and 3.0 mg L⁻¹ 6-BA.⁶⁸ Finally, spraying the leaves with a solution of Cu²⁺ and Zn²⁺ appears to stimulate the production of tanshinones in the root.⁶⁰

4. The mechanism of elicitor-induced tanshinone accumulation in dānshēn

The heightened accumulation of tanshinones induced by exogenously supplied MJ involves the activity of at least six enzymes, namely SmAACT, SmHMGS, SmHMGR, SmDXR, SmDXS2, SmGGPPS, SmIPPI and SmCPS. A slightly different group of enzymes – SmHMGR, SmDXS2, SmIPPI, SmFPPS, SmGGPPS and SmCPS – is important for enhancement in tanshinone content triggered by exposure to Ag⁺. The beneficial effect of supplementation with yeast extract involves the six enzymes SmHMGS, SmDXR, SmDXS2, SmCMK, SmIPPI and SmCPS, while the combination of yeast extract supplementation and Ag⁺ pre-treatment up-regulates the eight genes SmAACT, SmHMGS, SmDXR, SmDXS2, SmCMK, SmFPPS, SmGGPPS and SmCPS, and at the same time maintains a consistently high abundance of SmHMGR and SmIPPI transcript.⁴³ Yang et al.⁷¹ have shown that the abundance of SmHMGR, SmDXS and SmDXR transcripts, and the activity of SmHMGR and SmDXS are both stimulated by the presence of PEG, ABA and MJ. The genes SmHMGR and SmDXR are both up-regulated in the presence of nitric oxide,⁶³ SmHMGR, SmDXR, SmGGPPS, SmCPS and SmKSL are up-regulated by supplementation with the polysaccharide fraction of the endophytic fungus Trichoderma atroviride D16.⁵⁵ Thus, both the MVA and MEP pathways are activated by MJ, yeast extract, Ag⁺, PEG, ABA and nitric oxide, and as a result, so is tanshinone biosynthesis. It has been proposed that the exogenous supply of PEG and ABA triggers the release of endogenous MJ via the activation of an ABA signaling pathway and that this additional MJ (and similarly exogenously supplied MJ) provides the signal directing the increased production of tanshinones via the MEP pathway.⁷¹ Ge and Wu have suggested that the induction in tanshinone accumulation is response to supply of yeast extract plus Ag⁺ results from increased flux through the MEP pathway,⁴⁹ but that it may also involve an element of crosstalk with the MVA pathway, which is known to be an important determinant of cell growth.⁷⁹ Cu²⁺, Zn²⁺, MJ, PEG and ABA-induced tanshinone production is ROS-mediated, whereas that induced by nitric oxide is ROS-independent.^63,65,70

5. Metabolic engineering of tanshinone biosynthesis

The over-expression of SmHMGR2 increases the level of SmHMGR2 activity and enhances the production of tanshinones and squalene in cultured hairy roots,³⁸ while similarly, the over-expression of SmGGPPS and/or SmHMGR and/or SmDXS increases tanshinone production.⁸⁰ Of the three enzymes involved, SmGGPPS has the greatest effect on tanshinone production and SmHMGR the least. Simultaneously over-expressing SmHMGR and SmGGPPS results in a particularly high level of tanshinone production. The over-expression of SmAOC significantly enhances the yield of TIIA.⁸¹ Zhou et al.⁴⁴ have proposed a modular pathway engineering strategy to assemble a heterologous miltiradiene pathway in yeast. Miltiradiene is the precursor of tanshinones in dānshēn (Fig. 2). Fusion of SmCPS and SmKSL, and also of BTS1 (encoding GGPP synthase) and ERG20 (FPP synthase) significantly improves the yield of miltiradiene. The best performing transgenic yeast strain proved able to generate 365 mg L⁻¹ miltiradiene. Guo et al.²⁹ have further shown that the incorporation of genes encoding CYP76AH1 and phyto-CYP reductase in miltiradiene-producing yeast results in measurable amounts of ferruginol.

6. Conclusions and future prospects

The mechanistic basis of tanshinone biosynthesis has not yet been fully elucidated. The genes encoding a number of enzymes, included in this pathway in particular DXS, HDR, AACT, HMGR, GGPPS, CPS and KSL are members of multi-gene families, and these genes exhibit different patterns of expression in time and space, underlining the complexity of terpenoid biosynthesis in dānshēn. Different isoenzymes of one or all of these enzymes may be involved in the biosynthesis of specific terpenoids.²⁵ The later steps in tanshinone biosynthesis remain particularly obscure, so a current research priority is to identify which enzymes catalyse these steps. As yet, the identity of the transcription factors involved in the regulation of tanshinone biosynthesis has not been ascertained, although it is known that such factors represent a critical element in the biosynthesis of terpenoids in other species. For example, the Arabidopsis thaliana MYC2 protein, which belongs to the family of basic helix-loop-helix transcription factors, binds to the promoters of the sesquiterpene synthase genes TPS21 and TPS11.⁸² Similarly in tobacco the binding of an AP/ERF transcription factor to the promoter of a putrescine N-methyltransferase gene regulates its MJ-induced transcription and thereby influences the accumulation of nicotine and total alkaloids.⁸³ A key future line of research in dānshēn will therefore be the exploration of the identity and role of transcription factors in tanshinone biosynthesis. A combination of large-scale transcriptome sequencing and co-expression analysis, as demonstrated in Catharanthus roseus by Góngora-Castillo et al.,⁸⁴ provides a model strategy for exploring the regulation of tanshinone biosynthesis in dānshēn. The combined heterologous expression of the flavonoid activator transcription factor AtMYB12 and the legume isoflavone synthase gene IFS has been shown to promote the biosynthesis of isoflavone in tobacco leaves, even though the quantity of substrate present is unlikely to be sufficient.⁸⁵ Therefore the over-expression of both structural and regulatory genes, together with the suppression of side-branch enzyme genes, could represent a viable means of improving tanshinone production in dānshēn. Given that the tanshinone precursors miltiradiene and ferruginol have both been synthesized heterologously in yeast,^29,44 it should also be possible to produce tanshinone heterologously in well-developed, rapidly growing, high biomass-producing crop species such as tobacco and tomato. All the elicitors and treatments in this paper have positive effect on tanshinone biosynthesis. Combining any of yeast extract, fungal elicitor, sucrose, AIB, BABA, sorbitol, Co²⁺, Cu²⁺, Zn²⁺, Ag⁺, PEG, MJ, ABA, IAA, GA, NAA and 6-BA with one another has a larger enhancing effect than would be predicted from the effect each induces on its own.^{49,50,52–54,60,61,67,68,72} Cu²⁺, Zn²⁺, MJ, PEG and ABA all trigger a burst of ROS, which serves to raise the level of tanshinone production.^63,65,70 Yang et al.⁷¹ have shown that the crosstalk between PEG and ABA signalling pathways also has a regulatory effect on tanshinone biosynthesis. The variable effect on tanshinone accumulation and biomass growth of different elicitors and of the culture medium composition indicates that an effort needs to make to clarify the regulatory mechanisms underlying tanshinone biosynthesis.⁸⁶ A combination of metabolic engineering and elicitor treatments has the potential to support the sustainable production of tanshinone in the future.^26,43

Abbreviations

HIV-1	Human immunodeficiency virus type 1
HBV	Hepatitis B virus
TI	Tanshinone I
TIIA	Tanshinone IIA
TIIB	Tanshinone IIB
CT	Cryptotanshinone
DTI	Dihydrotanshinone I
EST	Expressed sequence tag
TDFs	Transcript-derived fragments
IPP	Isopentenyl diphosphate
DMAPP	Dimethylallyl diphosphate
MVA	Mevalonate
MEP	2-C-methyl-D-erythritol-4-phosphate
AACT	Acetyl-CoA C-acetyltransferase
HMGS	3-Hydroxy-3-methylglutaryl-CoA synthase
HMG-CoA	3-Hydroxy-3-methylglutaryl-CoA
HMGR	3-Hydroxy-3-methylglutaryl-CoA reductase
IPP	Isopentenyl diphosphate
MK	Mevalonate kinase
PMK	5-Phosphomevalonate kinase
MDC	Mevalonate 5-diphosphate decarboxylase
GA-3P	Glyceraldehyde 3-phosphate
DXP	1-Deoxy-D-xylulose 5-phosphate
DXS	DXP synthase
DXR	DXP reductoisomerase
MCT	Cytidyl transferase
CMK	4-(Cytidine 5-diphospho)-2-C-methyl-D-erythritol kinase
MDC	2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase
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 deltaisomerase
GPPS	Geranyl diphosphate synthase
GPP	Geranyl pyrophosphate
FPP	Farnesyl diphosphate
FPPS	Farnesyl diphosphate synthase
GGPPS	Geranylgeranyl diphosphate synthase
GGPP	Geranylgeranyl diphosphate
CPS	Copalyl diphosphate synthase
CPP	Copalyl diphosphate
KSL	ent-Kaurene synthase-like
SNP	Sodium nitroprusside
NO	Nitrogen oxide
MJ	Methyl jasmonate
IAA	Indol-yl-3-acetic acid
NAA	1-Naphthaleneacetic acid
GA3	Gibberellic acid
6-BA	6-Benzylaminopurine
TDZ	Thidiazuron
ABA	Abscisic acid
BABA	β-Aminobutyric acid
AIB	α-Animo isobutyric acid
PEG	Polyethylene glycol
ROS	Reactive oxygen species

Acknowledgements

This investigation was supported by Chinese Universities Scientific Fund (QN2013035 and 2014YB044), Natural Science Basic Research Plan in Shaanxi Province of China (2013JQ3006), Specialized Research Fund for the Doctoral Program of Higher Education (20130204120018 and 20130204120038), National Natural Science Foundation of China (81393708, 31401839 and 31170274), the Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant ‘Understanding and Exploiting Plant and Microbial Secondary Metabolism’ (BB/J004596/1), the John Innes Foundation, a joint Engineering and Physical Sciences Research Council/National Science Foundation award to AO as part of the Syntegron consortium (EP/K03459/1), and the EU FP7-funded programme Triterpenes for Commercialisation (TriForC). We thank Dr Ramesha B. Thimmappa for critical reading of the manuscript.

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