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

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

Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures

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

Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.     

Effects of ABA on primary terpenoids and Δ<Superscript>9</Superscript>-tetrahydrocannabinol in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. at flowering stage

This work examined the effects of exogenously applied abscisic acid (ABA) on the content of chlorophyll, carotenoids, α-tocopherol, squalene, phytosterols, Δ⁹-tetrahydrocannabinol (THC) concentration, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity in Cannabis sativa L. at flowering stage. Treatment with 1 and 10 mg l⁻¹ ABA significantly decreased the contents of chlorophyll, carotenoids, squalene, stigmasterol, sitosterol, and HMGR activity in female cannabis plants. ABA caused an increase in α-tocopherol content and DXS activity in leaves and THC concentration in leaves and flowers of female plants. Chlorophyll content decreased with 10 mg l⁻¹ ABA in male plants. Treatment with 1 and 10 mg l⁻¹ ABA showed a decrease in HMGR activity, squalene, stigmasterol, and sitosterol contents in leaves but an increase in THC content of leaves and flowers in male plants. The results suggest that ABA can induce biosynthesis of 2-methyl-d-erythritol-4-phosphate (MEP) pathway secondary metabolites accumulation (α-tocopherol and THC) and down regulated biosynthesis of terpenoid primary metabolites from MEP and mevalonate (MVA) pathways (chlorophyll, carotenoids, and phytosterols) in Cannabis sativa.     

Effects of Gibberellic Acid on Primary Terpenoids and △9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration,and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP)pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA3) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA3 caused a decrease in DXS activity in both sexes that it wasaccompanied by a decrease in chlorophylls, carotenoids and △9-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA3 showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants.The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA3 can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

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

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

Biosynthesis of the iridoid glucoside,lamalbid, in Lamium barbatum

The biosynthesis of the iridoid glucoside lamalbid in Lamium barbatum, a plant species in the Lamiaceae, was investigated by administrating ¹³C-labeled intermediates of MVA and MEP pathways, respectively. The results demonstrated that [3,4,5-¹³C₃]1-deoxy-d-xylulose 5-phosphate could be incorporated into lamalbid, whereas the incorporation of [2-¹³C₁]mevalonolactone was not observed. Based on the ¹³C labeling pattern of lamalbid and the incorporation data, we deduce that the iridoid glucoside in L. barbatum is biosynthesized through the MEP pathway, whereas the classic MVA pathway is not utilized.     

1-脱氧木酮糖-5-磷酸合成酶(DXS)及其编码基因

萜类物质是广泛分布于生物界的一类天然产物,也是重要生命物质。萜类物质通过甲羟戊酸(MVA)途径和2-C-甲基-D-赤藻糖醇-4-磷酸(MEP)途径合成,古细菌、真菌和动物及人的萜类物质主要通过MVA途径合成,而多数真细菌(即通常而言的细菌)则利用MEP途径。植物同时拥有两种途径但分别定位于细胞质和质体。1-脱氧木酮糖-5-磷酸合成酶(DXS)是MEP途径的第一个酶,也是该途径的关键调控位点。现从DXS在MEP途径中的作用、DXS结构、亚细胞定位和酶活性、编码基因及突变体等方面对DXS进行全面阐述。拟南芥DXS基因插入突变体cla1-1发生白化,DXS基因表达与类胡萝卜素等萜类物质积累密切相关,在转基因生物体中过度表达DXS可促进萜类物质合成。植物DXS具有典型的质体转运肽序列,决定了DXS的质体定位。完备的DXS活性分析体系为DXS抑制剂开发筛选等研究奠定良好基础。DXS由一至多个基因编码,随生物种类而异,根据同源性,植物DXS基因可分成两类。DXS基因家族不同成员具有不同的表达模式,但通常有一个成员在多种组织中广泛表达。     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

Optimized enzymatic preparation of 1-deoxy-d-xylulose 5-phosphate

The preparation of 1-deoxy-d-xylulose 5-phosphate, the key intermediate of MEP biosynthetic pathway for terpenoids by using recombinant 1-deoxy-d-xylulose 5-phosphate synthase of Rhodobacter capsulatus was optimized. The simple one-pot synthesis coupling with a newly established ion-exchange purification process affords the target compound with more than 80% yield and high purity (>95%). The procedure can also be employed to synthesize isotope labeled 1-deoxy-d-xylulose 5-phosphate by using isotope labeled starting materials.     

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-¹³C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.     

Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.     

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.     

不同花期‘西伯利亚’百合花瓣单萜合成途径转录组分析

以‘西伯利亚’百合（Lilium ‘Siberia’）花蕾期、半开期、盛开期、衰败期的花瓣为材料,利用RNA-seq技术对其转录组进行高通量测序,分析单萜合成途径中差异表达的基因并阐明其分子机制。结果显示,‘西伯利亚’百合通过转录组测序分析共得到56.28 Gb clean base,223.40 Mb clean reads和124 233个unigene,其中35 749个基因得到注释。萜骨架合成途径中的基因表达水平在不同花期表现出显著差异。其中,甲基赤藓糖醇磷酸（MEP）中的1-脱氧-D-木酮糖-5-磷酸合成酶（DXS）、1-脱氧-D-木酮糖-5-磷酸还原异构酶（DXR）、4-羟基-3-甲丁-2-烯基二磷酸合成酶（HDS）、4-羟基-3-甲丁-2烯基二磷酸还原酶（HDR）、牻牛儿基二磷酸合成酶（GPS）基因的表达水平随花期变化呈先升高后降低的趋势。罗勒烯合成酶（OCS）基因表现出相似变化规律,在盛开期表达量最高。甲羟戊酸（MVA）途径中的3-羟基-3-甲基戊二酸单酰辅酶A还原酶（HMGR）的基因表达同样出现先升高后降低的趋势。单萜合成下游的分支途径中,茄尼基二磷酸合成酶（SDS）、牻牛儿基牻牛儿基二磷酸合成酶（GGDR）基因的表达则出现相反的趋势,在盛开期的表达量最低。研究结果表明MEP途径中的关键基因可随花期变化规律性的表达,以调控单萜的生物合成,在盛开期有较高释放量,且盛开期MVA途径的活化以及泛醌和萜醌代谢支路基因的低表达也促进了单萜的生物合成。