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.     

Hairy roots of Datura candida×D. aurea: effect of culture medium composition on growth and alkaloid biosynthesis

A transformed root clone of Datura candida×D. aurea was established following infection with Agrobacterium rhizogenes strain A4. This clone was examined for its growth and hyoscyamine and scopolamine content under various culture conditions. Among the three basal culture media tested, half-strength Gamborg's B5 medium supplemented with 5% sucrose was the best for root growth (288 mg dry weight/flask) and full-strength B5 medium for hyoscyamine and scopolamine content (0.36 and 0.17% dry weight, respectively). Experiments with exogenous nitrate added to the medium revealed that the biomass increased (353 mg dry weight/flask) and the hyoscyamine content improved remarkably (0.54% dry weight), but that the scopolamine content was significantly reduced. The addition of various precursors at two different concentrations did not significantly modify root growth. Feeding (R,S)-phenyllactic acid stimulated the biosynthesis of both alkaloids, whereas the addition of ornithine specifically reduced the scopolamine content. Received: 12 March 1997 / Revision received: 22 April 1997 / Accepted: 5 May 1997     

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D₂ ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.     

Production of Tropane Alkaloids by Hairy Root Cultures of Scopolia japonica

Hairy root clones of Scopolia japonica were established by selection of adventitious roots formed on the root segments inoculated with Agrobacterium rhizogenes strain 15834. Twenty-nine isolated hairy root clones displayed various phenotypes characterized by growth rate, opine production and tropane alkaloid production. Of these, two highly alkaloid productive clones SI and S22 were examined for their growth rate and alkaloid productivity under various cultural conditions. When the most scopolamine-productive clone SI was cultured for 4 weeks at 25°C in the dark, the weight of the root tissue was increased by 40 times and the content of scopolamine reached a level of 0.5% on a dry weight basis in each optimum medium. On culture of the most hyoscyamine-productive clone S22 under the same conditions as with S1, the weight was increased by 102 times and the content of hyoscyamine was 1.3% on a dry weight basis in each optimum medium.     

Effect of phosphate on ergot alkaloid synthesis inAspergillus fumigatus

High concentration of inorganic phosphate in the culture medium ofAspergillus fumigatus inhibited ergot alkaloid synthesis. Addition ofl-tryptophan but not mevalonate or 5-methyltryptophan to the above culture restored the alkaloid synthesis to the level found in normal cultures. The decrease in alkaloid synthesis in the fungus accompanies an increase in cell mass, cellular protein and sterol content. Aspartate aminotransferase and alanine aminotransferase activities were significantly increased in the high-phosphate culture. Part of the work was presented at the seminar on “Enzymatic Methods in Mycology” organised by the Czechoslovak Microbiological Society in Brno, Czechoslovakia, in June 1975.     

Influence of the ion-composition of the medium on alkaloid production by “hairy roots” ofDatura stramonium

The influence of culture age on biomass production and alkaloid yield of “hairy roots” obtained after infection ofDatura stramonium L. withAgrobacterium ATCC 15834 was investigated. Maximal hyoscyamine yield was obtained with roots harvested after six weeks. Fluctuations were found for tropine yield, the precursor of the ring moiety of hyoscyamine. These indicate a continuous conversion to hyoscyamine during the exponential growth phase. The effect of the ion-balance was investigated by preparing five different media that only differed in their ionic composition. The ionic interactions between macroelements, differently influenced biomass production and alkaloid yield. As a result, highest biomass yield was found with NO₃ ⁻ - and K⁺-dominance, whereas hyoscyamine yield was highest with the culture medium in which SO₄ ²⁻ and K⁺ were dominant. Shifting the intercationic balance to strongly towards Ca²⁺ caused an overall reduced metabolism, since as well biomass yield as hyoscyamine yield was lowest with the NO₃ ⁻Ca²⁺-medium. Also tropine yield was affected by the ion-balance, indicating that this culture parameter also influences alkaloid synthesis.[/p]     

Influence of inorganic phosphate and immobilization on Claviceps purpurea

Summary The influence of inorganic phosphate and immobilization on cells of Claviceps purpurea strain 1029/N5 producing ergot peptides in shake culture was examined. Immobilization in Ca-alginate beads resulted in a marked reduction of some metabolic activities, i.e. the periods of alkaloid formation and cell growth were prolonged. High concentrations of inorganic phosphate (1 g/l KH₂PO₄) could reduce or stop alkaloid formation both by free and immobilized cells at any time during fermentation. The optimum phosphate concentration for alkaloid production by immobilized cells (about 0.5 mM) was a quarter of that required by free cells. This optimum shift was attributed to (i) the diminished phosphate demand of immobilized cells, due to their reduced metabolic activities, and (ii) the phosphate-dependent morphological behaviour of the biocatalyst. The observed decrease in alkaloid concentrations during later periods of the fermentation supported the idea of alkaloid-degradative enzymes, activated by high phosphate concentrations. Immobilization showed an advantageous influence on this undesirable effect. Offprint requests to: H.J. Rehm     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Inducer effect of Tween 20 permeabilization treatment used for release of stored tropane alkaloids inDatura innoxia Mill. hairy root cultures

Summary The effects of Tween 20 as permeabilizing agent on tropane alkaloids fromDatura innoxia Mill. hairy root cultures have been studied. The kinetics of the alkaloid release is detailed and shows three different stages: an initial rapid increase of the alkaloid level within the roots and in the culture medium, followed by a slower but higher increase of the alkaloid concentration in the medium. During this phase, the alkaloid concentration within the roots returned to a lower value. Finally, after a longer time, the quantity of hyoscyamine in the medium decreased significantly with a variable rate. According to the total alkaloid content per flask determinations under different conditions, it is clearly demonstrated that Tween treatment permeabilized the roots, but also acted as an inducer.     

A preliminary evaluation of the use of microbial culture filtrates for the control of contaminants in plant tissue culture systems

The partitioning of carbon between reserve polysaccharide and alkaloid secondary products was investigated in batch cultures of transformed roots of Datura stramonium grown in media in which the carbon substrate concentration was held constant and the level of mineral nutrients was varied. The growth and accumulation of starch and hyoscyamine was examined in roots grown at temperatures of 20°C, 25°C or 30°C in media containing 5% sucrose and levels of mineral nutrients varying from 1/4 to twice the standard level of Gamborg's B5 salts. The dry matter content was highest (up to 15% w/w) in roots grown at either 20°C or 25°C in medium of the lowest ionic strenth (1/4 B5 salts) and decreased as the ionic strength was raised (down to 7% w/w with 2 B5 salts). Up to half of this decrease could be accounted for by loss of starch from the roots. At 20°C and 25°C, the starch content of the roots grown in medium of the lowest ionic strength (1/4 B5) was 40 mg g^-1 and 22 mg g^-1 fresh weight respectively but decreased to less than 1 mg g^-1 weight at either temperature when the ionic strength of the medium was raised to 2 B5. At 30°C, starch accumulation was severely inhibited in all media. In contrast, varying either the temperature or the ionic strength of the medium had only a small effect on hyoscyamine accumulation which remained at between 0.4–0.6 mg g^-1 fresh weight. Although increases in the level of mineral salts had little effect on the hyoscyamine content of the roots, total yields however, increased due to stimulation of growth. Time course experiments showed that cultures grown at either 20°C or 25°C continued to accumulate both starch and hyoscyamine into late stationary phase.     

Tailoring Tropane Alkaloid Accumulation in Transgenic Hairy Roots of Atropa baetica by Over-expressing the Gene Encoding Hyoscyamine 6β-hydroxylase

Atropa baetica hairy roots, over-expressing cDNA from Hyoscyamus niger encoding the gene for hyoscyamine 6β-hydroxylase (H6H), were produced by Agrobacterium rhizogenes infection. The transgenic roots over-expressing h6h had an altered alkaloid profile in which hyoscyamine was entirely converted into scopolamine. In the best h6h clone, scopolamine accumulation increased 9-fold compared to plants, amounting to 5.6 mg g dry wt⁻¹, some of which was released into the liquid medium. Only negligible amounts of hyoscyamine were detected. In contrast, the gus control culture contained a much higher amount of hyoscyamine than scopolamine, mimicking the situation in the plant. At the molecular level, a higher conversion of hyoscyamine into scopolamine was related to a higher level of h6h mRNA; in some instances this was 5–10-fold higherThis article is dedicated to the memory of Professor Antonio G. González     

Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin

A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, strictosidine, 10-hydroxy strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.     

Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus

Hyoscyamus muticus accession was evaluated for its response to inoculation with Agrobacterium rhizogenes strains LBA9402, A4, 15834, and Agrobacterium tumefaciens strain C58 CI pRT GUS104. The Agrobacterium strain used for the transformation has a significant influence on the phenotype of the clone as well as on the growth rate and hyoscyamine production of these root culture clones. The most virulent strains were C58 CI pRT GUS104 and LBA9402. More roots were obtained on LSO medium than on B50 medium. Acetosyringone addition and the time from wounding affected root formation. The alkaloid content was highest in clones C58 and A4 (90mg·l^-1). There are great differences between individual hairy root clones, and hence they are not as uniform as has often been speculated. The Agrobacterium strain used for the transformation has a great influence in this respect.     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

Growth and anthocyanin production by carrot suspension cultures grown under chemostat conditions with phosphate as the limiting nutrient

Chemostat cultures of carrot suspension cultures, where growth was limited by the concentration of phosphate in the input medium, were achieved by replacing a fixed proportion of the culture with fresh medium at daily intervals. In the range 0.05–0.30mM phosphate in the input medium and at a specific growth rate of 0.357 days^?1, steady-state culture density but not anthocyanin in the cells was strictly proportional to the input phosphate concentration with no intercept. At a phosphate concentration of 0.10mM and growth rates from 0.105 to 0.430 days^?1, the steady-state culture density could not be described by Monod's model of chemostat cultures, but could be described by Nyholm's model. The steady-state levels of anthocyanin were not strictly proportional to the steady-state biomass under all conditions, showing that anthocyanin production is not completely growth associated.     

Effects of calcium,alginate, and calcium-alginate immobilization on growth and tropane alkaloid levels of a stable suspension cell line of Datura innoxia Mill

Summary A stabilized two-year old suspension of a Datura innoxia cell line, producing small amounts of tropane alkaloids (scopolamine and hyoscyamine) was used in this study. Calcium alginate immobilization has been shown to be able to increase secondary metabolite (i. e. alkaloid) production. The effects of calcium and ungellified alginate were both beneficial for tropane alkaloid synthesis; a 10mM calcium chloride supply gave the best results, with a 10-fold yield increase.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - S standard cell culture medium - HPLC high performance liquid chromatography - FW fresh weight - FDA fluorescein diacetate - FW fresh weight     

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.     

A study on the alkaloid production by resting cell suspensions of Catharanthus roseus in a continuous flow reactor

Summary During batch cultivation of Catharanthus roseus cell suspensions, alkaloids were found in the culture medium after growth had ceased. Resting cell suspensions with high alkaloid content were obtained by transferring the cells to a medium devoid of 2.4 D (2.4 dichlorophenoxy-acetic acid). A production system with continuous feeding was developped to study alkaloid production by these resting cell suspensions.     

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.     

Variation of alkaloid productivity among several clones of hairy roots and regenerated plants ofAtropa belladonna transformed withAgrobacterium rhizogenes 15834

Hairy root cultures ofAtropabelladonna were established by transformation withAgrobacterium rhizogenes 15834. Five clones of them were employed to study the production of hyoscyamine, the main constituent of the plant, together with other tropane alkaloids. The growth and alkaloid production of each clone were differently affected by basal liquid culture media tested. The transgenic plants regenerated from each clone of the hairy roots had different phenotypes and diverse alkaloid productivity both in the cultured condition and in productivitiy both in the cultured condition and in hydroponics.Abbreviations ANOVA analysis of variance - B5 medium Gamborg B5 medium - BA N⁶-benzyladenine - B.S. Balanced Solution - dw dry weight - EC electric conductivity - fw fresh weight - GC/MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MS medium Murashige and Skoog medium - NAA naphthalene-l-acetic acid - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TMS trimethylsilyl - WP medium Woody Plant medium