Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.     

Transient effects of overexpressing anthranilate synthase alpha and beta subunits in Catharanthus roseus hairy roots

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase alpha subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase beta subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, h?rhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.     

Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum

Silymarin is one of the most potent antioxidant so far developed from plant sources used as hepatoprotectants. Influence of different concentrations (0, 1, 2, 4, 6 and 8 mg/50 ml culture) and exposure time (24, 48, 72, 96 and 120 h) of salicylic acid on lipoxygenase activity, linoleic acid content, growth and production of silymarin in hairy root cultures of S. marianum were investigated. Detection and identification of flavonolignans was carried out by high performance liquid chromatograph method. Salicylic acid enhanced silymarin production (1.89 mg g⁻¹ DW). The optimal feeding condition was the addition of salicylic acid (6 mg/50 ml culture) after 24 h in which the silymarin content was 2.42 times higher than the control (0.78 mg g⁻¹ DW). The content of silybin, isosilybin, silychristin, silydianin and taxifolin were 0.703, 0.017, 0.289, 0.02 and 0.863 mg g⁻¹ DW respectively in these samples, while in non-treated hairy roots were 0.027, 0.046, 0.23, 0.022 and 0.453 respectively. Lipoxygenase activity also affected by elicitation. lipoxygenase activity increased 24 h after treatment by ∼1.57- fold (0.21 Δ OD₂₃₄/mg protein min⁻¹). Upon elicitation with salicylic acid, linoleic acid content of hairy roots (38.26 mg g⁻¹ DW) were also elevated after 24 h, in which the linoleic acid content was 2.37 times higher than the control (16.1 mg g⁻¹ DW). It is feasible that elicitation with salicylic acid regulates the jasmonate pathway, which in turn mediates the elicitor-induced accumulation of silymarin.     

Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.     

Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.     

Overexpression of a chrysanthemum transcription factor gene <Emphasis Type="Italic">DgNAC1</Emphasis> improves drought tolerance in chrysanthemum

Phenolic acids and tanshinones are two groups of pharmaceutical components present in Salvia miltiorrhiza Bunge. Methyl jasmonate (MeJA) has been reported to influence the accumulation of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. However, there is currently a lack of information regarding the comparison of how these two groups of bioactive compounds in S. miltiorrhiza respond to MeJA under the same conditions. In the present study, the effect of 100 µM MeJA on the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza hairy roots was investigated. The results showed that MeJA dramatically induced the accumulation of five different phenolic acids, especially rosmarinic acid and salvianolic acid B, which reached their highest contents at day 3 (20.3 mg/g DW, 1.5-fold of control) and day 6 (47.49 mg/g DW, 2.5-fold of control), respectively. The total production of phenolic acids was induced by as much as 3.3-fold of the control (day 9 after treatment), reaching 357.5 mg/L at day 6. However, tanshinone I was almost unaffected by MeJA treatment, and the accumulation of tanshinone IIA was inhibited. Furthermore, cryptotanshinone and dihydrotanshinone I were moderately induced by MeJA. The gene expression results indicated that MeJA probably induced the whole pathways, especially the tyrosine-derived pathway and the methylerythritol phosphate pathway, and finally resulted in the increased production of these metabolites. This study will help us to further understand how the different biosynthetic mechanisms of phenolic acids and tanshinones respond to MeJA and provide a reference for the future selection of elicitors for application to improving the production of targeted compounds.     

Characterization of an inducible promoter system in Catharanthus roseus hairy roots

Transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of green fluorescent protein (GFP), and GFP expression was characterized. The inducible system shows a tightly controlled, reversible, and dosage-dependent response to the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was noted after 12-18 h in the mature regions of the root tips and after 6 h in the meristem tissue. Upon removal of the inducing agent, GFP expression declined to undetectable levels in the mature tissues after 24 h and in the meristem after 48 h. Although no dosage-dependent response was noted in the meristem region, such a response was apparent in the mature region of the tip and verified by quantitative GFP analysis. The inducible promoter system allowed quantitative control of GFP expression between 0.01 and 10 microM dexamethasone with saturation occurring at higher levels. Using GFP as a model system allowed demonstration of the ability to control temporal and quantitative gene expression with the glucocorticoid-inducible promoter in transgenic C. roseus hairy roots.     

Transient studies of nutrient uptake, growth, and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus

The kinetics of growth, the uptake of macronutrients, and the accumulation of indole alkaloids were investigated in long-term, heterotrophically cultured transgenic ("hairy") roots of Catharanthus roseus.Tabersonine, ajmalicine, and serpentine were monitored over a 70-day period. The doubling time [dry-weight (DW) basis] of C. roseus hairy roots in B5/2 nutrients supplemented with 3% sucrose was 3.6 days. NH(4) (+), NO(3),(-) and P(i) were depleted sequentially from culture medium by hairy roots, while sugars remained undepleted. The growth-limiting nutrient was inorganic nitrogen, NH(4) (+) and NO(3) (-), with exponential-phase overall biomass yields of 34.1 and 5.0 g DW/g nutrient, respectively. Extracellular pH decreased to 4.8 in early exponential phase of culture growth from the initially adjusted value of 5.7, increased subsequently to a maximum of 7.7 in late exponential phase of growth coincident with the maximum of fresh weight (FW)/DW ratio, before decreasing to 5.5-5.0. The organic acids, pyruvate, formate, lactate, and succinate were excreted by hairy roots starting in late phase of exponential growth, possibly resulting in the late-culture pH decrease. Tabersonine accumulation was distinctly growth associated with maximum specific and total yields of 1.15 mg/g DW and 5.6 mg/L, respectively, in late-exponential phase of growth. Serpentine accumulation was non growth associated with increasing specific and total levels in stationary growth phase: 1.3 mg/g DW and 10.5 mg/L, respectively. The accumulation of ajmalicine also appeared growth associated. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 527-534, 1997.     

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in h?rhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.     

Inhibitor studies of tabersonine metabolism in C. roseus hairy roots.

The conversion of tabersonine to lochnericine and h?rhammericine was investigated in C. roseus hairy root cultures. The accumulation of lochnericine and h?rhammericine, like tabersonine, was associated with growth. Through the use of oxygenase inhibitors, 1-aminobenzotriazole (ABT), clotrimazole (CLOT), and 2.5-pyridinedicarboxylic acid (PCA), details of the metabolic pathway around tabersonine in hairy roots of C. roseus were elucidated. ABT specifically inhibited the formation of h?rhammericine, while CLOT inhibited the accumulation of lochnericine. Using jasmonic acid in combination with the inhibitors suggests an inducible P-450 enzyme responsible for the formation of h?rhammericine. The inhibitor study also revealed that both lochnericine and h?rhammericine are 'turned over' in hairy root cultures.     

Differential Secretion and Accumulation of Terpene Indole Alkaloids in Hairy Roots of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> Treated with Methyl Jasmonate

The induction of several secondary metabolites in plants is one of the most commonly observed effects after the external addition of methyl jasmonate (MeJA). After the elicitation of Catharanthus roseus hairy roots with different concentrations of MeJA, changes in the accumulation of alkaloids such as ajmalicine, serpentine, ajmaline and catharanthine were observed. In addition to the increased accumulation of alkaloids in the tissues, the root exudation of phytochemicals increased compared to that of the non-treated control hairy roots. Moreover, MeJA induced differential secretion of several C. roseus hairy root metabolites.     

Salicylic acid‐enhanced biosynthesis of pharmacologically important lignans and neo lignans in cell suspension culture of Linum ussitatsimum L

Linum usitatsimum L. (flax) is a perennial herb with magnitude of medicinal and commercial applications. In the present study, we investigated the effects of salicylic acid (SA) on biosynthesis of lignans (secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG)) and neolignans (dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol‐β‐coniferyl alcohol ether glucoside (GGCG)) in cell cultures of flax. Moderate concentration of SA (50 μM) enhanced biomass accumulation (10.98 g/L dry weight (DW)), total phenolic content (37.81 mg/g DW), and antioxidant potential (87.23%) to two‐fold than their respective controls after 72 h of exposure. However, higher levels of total flavonoid content (5.32 mg/g DW) were noted after 48 h of exposure to 50 μM of SA. HPLC analyses revealed that 50 μM SA, significantly enhanced biosynthesis of SDG (7.95 mg/g DW), LDG (7.52 mg/g DW), DCG (54.90 mg/g DW), and GGCG (16.78 mg/g DW), which was almost 2.7, 1.8, 3.88, and 3.98 fold higher than their respective controls after 72 h of exposure time, respectively. These results indicated that moderate concentrations of SA had significant effects on biosynthesis and productivity of lignans and neolignans in cell culture of L. usitatissimum.     

Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.     

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.