Alkaloid spectrum in diploid and tetraploid hairy root cultures of Datura stramonium

Hairy root cultures were obtained from diploid and induced tetraploid plants of Datura stramonium and analyzed by gas chromatography/mass spectrometry. Twenty alkaloids (19 for diploid and 9 for tetraploid hairy root cultures) were identified. A new tropane ester 3-tigloyloxy-6-propionyloxy-7-hydroxytropane was identified on the basis of mass spectral data. Hyoscyamine was the main alkaloid in both diploid and tetraploid cultures. In contrast to diploid hairy roots, the percentage contributions of the alkaloids, with exceptions for hyoscyamine and apoatropine, were higher in the total alkaloid mixture of tetraploid hairy roots.     

Enhanced production of hyoscyamine and scopolamine from genetically transformed root culture of <Emphasis Type="Italic">Hyoscyamus reticulatus</Emphasis> L. elicited by iron oxide nanoparticles

The medicinal plant Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with Agrobacterium rhizogenes and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L^?1) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L^?1 FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L^?1 FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production.     

Establishment of hairy root cultures ofDatura stramonium. Characterization and stability of tropane alkaloid production during long periods of subculturing

The aim of this paper was the screening of the variability of growth patterns, biomass and tropane alkaloid production of 500 hairy root lines ofDatura stramonium. Data on the long term stability in alkaloid production of these lines for more than 5 years are also provided. In an effort to obtain high alkaloid-producing root clones, it is demonstrated that systematic selection is necessary. Comparisons are made, mainly concerning alkaloid production and its stability, with normal root cultures initiated from the same mother plants when necessary. Hairy root cultures were found to have a hyoscyamine and scopolamine bioproductivity of 2 orders of magnitude higher than mother plants.     

Effects of acetylsalicylic acid and UV-B on gene expression and tropane alkaloid biosynthesis in hairy root cultures of Anisodus luridus

Anisodus luridus hairy root cultures were established to test biological effects of acetylsalicylic acid (ASA) and ultraviolet ray-B (UV-B) on gene expression, tropane alkaloid (TA) biosynthesis and efflux. The TAs-pathway gene expression was ASA dosage dependant. The expression of PMT, TRI and CYP80F1 showed no significant difference in hairy root cultures in treatment of 0.01 and 0.1 mM ASA, compared with those without ASA treatment; while 0.01 or 0.1 mM ASA slightly upregulated H6H expression. All the four genes including PMT, TRI, CYP80F1 and H6H had a dramatic increase in 1 mM ASA-treated hairy root cultures compared with control. The expressing levels of all the four genes were much significantly higher in 1 mM ASA-treated hairy root cultures than those in 0.01 and 0.1 mM ASA-treated ones. As expected, hairy root cultures treated with 1 mM ASA had the highest capacity of TAs biosynthesis, in which the content of scopolamine and hyoscyamine reached respectively 57.2 and 14.7 μg g^?1 DW. Surprisingly, it was found that 1 mM ASA dramatically induced the efflux of scopolamine. In the liquid medium with 1 mM ASA, the content of scopolamine was 153.4 μg flask^?1, about 6.2 folds compared with that of control. At the same time, hyoscyamine was detected at trace levels in liquid medium. In the UV-B stressed hairy root cultures, all the four genes had a very strong increase of gene expression that led to more accumulation of scopolamine and lower accumulation of hyoscyamine. Only trace amounts of hyoscyamine and scopolamine were detected in the liquid medium when hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.     

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Norlittorine and norhyoscyamine identified as products of littorine and hyoscyamine metabolism by 13C-labeling in Datura innoxia hairy roots

The presence of two compounds, norlittorine and norhyoscyamine, has been reported in leaves and roots of Datura innoxia; however their metabolic origin in the tropane alkaloid pathway has remained unknown. Precise knowledge of this pathway is a necessary pre-requisite to optimize the production of hyoscyamine and scopolamine in D. innoxia hairy root cultures. The exact structure of norlittorine and norhyoscyamine was confirmed by LC–MS/MS and NMR analyses. Isotopic labeling experiments, using [1-¹³C]-phenylalanine, [1′-¹³C]-littorine and [1′-¹³C]-hyoscyamine, combined with elicitor treatments, using methyl jasmonate, coronalon and 1-aminocyclopropane-1-carboxylic acid, were used to investigate the metabolic origin of the N-demethylated tropane alkaloids. The results suggest that norlittorine and norhyoscyamine are induced under stress conditions by conversion of littorine and hyoscyamine. We propose the N-demethylation of tropane alkaloids as a mechanism to detoxify cells in overproducing conditions.     

Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering

 In order to test the possibility of enhancing the production of pharmaceutically valuable scopolamine in transgenic cultures, the 35S-h6h transgene that codes for the enzyme hyoscyamine-6β-hydroxylase (EC 1.14.11.11) was introduced into Hyoscyamus muticus L. strain Cairo (Egyptian henbane). This plant was chosen for its capability to produce very high amounts of tropane alkaloids (up to 6% of the dry weight in the leaves of mature plant). To our knowledge, this is the first time such a large population of transgenic cultures has been studied at the morphological, chemical and genetic levels. A great variation was observed in the tropane alkaloid production among the 43 positive transformants. The best clone (KB7) produced 17 mg/l scopolamine, which is over 100 times more than the control clones. However, conversion of hyoscyamine to scopolamine was still incomplete. The expression of h6h was found to be proportional to the scopolamine production, and was the main reason behind the variation in the scopolamine/hyoscyamine ratio in the hairy-root clones. These results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more efficient gene-expression systems. Received: 24 December 1998 / Accepted: 13 January 1999     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.     

Production of tropane alkaloids by small-scale bubble column bioreactor cultures of Scopolia parviflora adventitious roots

The mass production of tropane alkaloids from adventitious root cultures of Scopolia parviflora, in small-scale bubble column bioreactor (BCB) was attempted. Adventitious roots of S. parviflora produced relatively enhanced levels of scopolamine and hyoscyamine in bioreactor compared to flask type cultures, and rapidly produced root clumps, with continuously increasing biomass throughout the culture period. The production of scopolamine and hyoscyamine in the top and bottom regions of root clumps were higher than in the core region. The adventitious root cultures of S. parviflora in the BCB required a relatively high level of aeration. The optimized conditions for the bioreactor culture growth and alkaloid production were found to be 3g of inoculum, on a fresh weight basis, a 15-day culture period and 0.4vvm of airflow. The elicitation by Staphylococus aureus increased the specific compound of scopolamine, while the production of hyoscyamine was slightly inhibited in BCB cultures.     

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.     

High-yield production of tropane alkaloids by hairy-root cultures of aDatura candida hybrid

Hairy root cultures were obtained following inoculation of the stems of sterile plantlets of aDatura candida hybrid withAgrobacterium rhizogenes. The scopolamine and hyoscyamine content was quantified by HPLC and compared with the non-transformed plants. The alkaloid yield (0.68% dry weight) obtained with the hairy roots was 1.6 and 2.6 times the amount found in the aerial parts and in the roots of the parent plants, respectively. Only a small proportion of alkaloids was released into the growth medium. Scopclamine was the principal alkaloid and the scopolamine/hyoscyamine ratio of ca. 5:1 makes these hairy roct cultures worthy of consideration as a source of scopolamine.     

Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography

Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.     

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l ^-1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l ^-1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Scopolamine,anisodamine and hyoscyamine production by Brugmansia candida hairy root cultures in bioreactors

Hyoscyamine, anisodamine (6β-hydroxyhyoscyamine) and scopolamine are tropane alkaloids produced by plants belonging to the Solanaceae family such as Brugmansia candida. These alkaloids were traditionally used in medicine because of their anticholinergic activity. Further therapeutical properties for anisodamine were recently described renewing the interest in these alkaloids. The scaling-up of hairy root cultures is an interesting strategy for the pharmaceutical production of these compounds instead of the isolation from plants. In this work, B. candida hairy roots were cultured in a modified 1.5 L stirred tank in order to analyze an alternative production system of scopolamine and anisodamine. It was found that these cultures produced an increased biomass and alkaloids concentration compared to the processes carried out in Erlenmeyer flasks. Anisodamine was the predominant alkaloid reaching a maximum concentration of 10.05 ± 0.76 mg/g DW in modified bioreactor culture system. The results obtained in this work are potentially applicable for the rational scale-up of the process.     

Analysis of the Potential Use of Androgenic Datura innoxia for the Development of Cell Cultures Producing High Amounts of Tropane Alkaloids

Normal and androgenic diploid Datura innoxia plants were selfedand the progeny was analysed for its leaf alkaloid content.Since the androgenic lines had originally produced very differentamounts of the tropane alkaloids, scopolamine and hyoscyamine,we were interested in determining whether this trait is transmittedby self-fertilization. The alkaloid content of the progeny wasfound to correlate well with that of the parental plants. Also,calli were initiated from leaf discs derived from plants withdifferent capacities for alkaloid biosynthesis. These were furthersubcultured for 2 years. Again, the same correlations in hyoscyamineand scopolamine content were observed. This indicates that itis possible to initiate callus with a high alkaloid contentstarting from actively alkaloid-producing androgenic Daturainnoxia plants. Key words: Datura innoxia, tropane alkaloids, androgenic plants, callus culture     

Characterization of transgenic plants derived from hairy roots ofHyoscyamus muticus

Mature plants were regenerated via protoplasts fromAgrobacterium rhizogenes-transformed root cultures ofHyoscyamus muticus L., and chemical analyses were performed on 34 individual plants. The regenerated plants showed strong phenotypic differences from clone to clone as well as from the control plants. Polymerase chain reaction studies revealed that the plants exhibiting the strongest phenotypic alterations contained therol (A, B and C) genes, whereas the plants with fewer alterations had lost them. The plants produced hyoscyamine, scopolamine and a range of different calystegins, and considerable somaclonal variation was observed. Alkaloid production in the plants transgenic for therol genes was clearly reduced. The pattern of calystegins was similar within all the regenerated plants lackingrol genes. Among the plants withrol genes, the calystegin B₁ was not detectable. It seems clear that the presence ofrol genes is detrimental to the alkaloid accumulation in the transgenic plants in contrast to hairy root cultures.Abbreviation PCR Polymerase chain reaction     

Hyoscyamine and proline accumulation in water-stressed Hyoscyamus muticus ‘hairy root’ cultures

Summary The patterns of hyoscyamine and proline accumulation were studied in Agrobacterium-transformed ‘hairy root’ cultures of Hyoscyamus muticus to determine if proline is a metabolic precursor of hyoscyamine. Root cultures were stressed osmotically with mannitol and the subsequent growth, hyoscyamine levels, and proline levels were measured after each transfer to fresh experimental medium for a total of four transfers. H. muticus ‘hairy roots’ were also treated with [U-¹⁴C] proline or [1,4-¹⁴C] putrescine and analyzed for radioactive hyoscyamine. Growth of ‘hairy root’ cultures was reduced by up to 90% in 0.4 M mannitol, and this inhibition persisted for at least four transfers. ‘Hairy root’ cultures of H. muticus accumulated hyoscyamine and free proline (up to 6-fold and 25-fold, respectively) when osmotically stressed with mannitol, and this effect also persisted for four transfers when grown in the same mannitol concentration. Because the total production of hyoscyamine was also increased by twofold, we conclude that the elevated hyoscyamine concentration results from increased hyoscyamine synthesis and not from reduced growth. H. muticus ‘hairy roots’ incorporated radioactivity from [1,4-¹⁴C] putrescine efficiently into hyoscyamine in both treatments, but failed to convert [U-¹⁴C] proline into hyoscyamine. We thus conclude that accumulated proline does not serve as a precursor for hyoscyamine.