Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ,elicitors and alginate immobilization

Summary More efficient bioreactors for the production and recovery of secondary metabolites from plant cell cultures are needed. Three factors that have the potential to increase productivity are adsorption in situ, elicitors, and cell immobilization. The effects of these factors on ajmalicine production from Catharanthus roseus are reported in this paper. Elicitation using autoclaved cultures of the mold, Phytophthora cactorum, stimulates a 60% increase in ajmalicine production. The response time to elicitor addition was under 11 h. Adsorption of ajmalicine from the extracellular medium with the neutral resin, Amberlite XAD-7, greatly enhanced the release of ajmalicine (less than 10% extracellular to 40%) with a 40% increase in total productivity. Immobilization in Caalginate beads resulted in a significant increase in the accumulation of ajmalicine in the medium. The effects of elicitation, adsorption and immobilization were synergistic. For a 23-day culture period the amount of ajmalicine in the medium for cells subjected to all three treatments was 90 mg/L compared to 2 mg/L for suspension cultures cultured under otherwise identical conditions. These results suggest that immobilized cell bioreactors may be feasible for continuous production of products normally stored intracellularly in vacuoles in plant cells.     

Impact of hypoxia on the growth and alkaloid accumulation in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension

The Madagascar periwinkle (Catharanthus roseus) produces numerous indole alkaloids, several of which have an important pharmaceutical uses such as ajmalicine, vinblastine and vincristine. The relationship between hypoxia and ajmalicine production in a cell suspension culture of C. roseus were investigated during the cycle of cell culture, in correlation with the effects on growth. The results show that the lack of oxygenation in C20D cells provokes a very strong inhibition in accumulation of the alkaloids and of other possible substances. Moreover, the addition of loganin, a metabolic intermediate of the biosynthetic pathway, in the culture medium of cells subjected to hypoxia restored the alkaloid production. Also, the results showed that the addition of benzyladenine (BA) to the culture medium increased the ajmalicine production and that the inhibitory effect of hypoxia was almost absent in these conditions. Therefore, it could be suggested that BA can without doubt decrease the effects of the hypoxia and increase the ajmalicine production in periwinkle cell suspensions.     

Elicitor-stimulation of Monoterpene Indole Alkaloid Formation in Suspension Cultures of Catharanthus roseus

Upon treatment of 5 cell lines of Catharanthus roseus with homogenates of various fungi, as well as with chemically defined phytoalexin elicitors, all except one (non-alkaloid producing #916) responded with browing and accumulation of tryptamine within 6 – 24 h. Cells of line #615 responded with not only accumulating tryptamine, but also N-acetyl tryptamine, strictosidine lactam, ajmalicine, tabersonine, lochnericine, and catharanthine. Based on amounts of alkaloids accumulated, cells of line #615 performed best when treated with homogenates of Alternaria zinnae, Pythium apbanidermatum, Verticillium dabliae, and Rhodotorula rubs. A Pythium homogenate concentration of 5 % and a Rhodotorula homogenate concentration of 0.5 % effected maximum alkaloid yields, and, thus, were used in subsequent studies. These revealed a temporary increase of the level of alkaloids in cells and in their medium after 12 – 24 h of treatment. Ten-day-old subcultures responded better than younger and older ones. The elicitor stimulated accumulation of alkaloids and alkaloid composition did not depend on the use of 1-B5 or alkaloid production medium. A 5 l cell suspension of #615 grown in a 7.5 l bioreactor and treated with 5 % Pythium homogenate for 18 h was found to contain strictosidine lactam, ajmalicine, and catharanthine in concentrations of 27, 10, and 13 μg/g DW respectively, the medium contained 42 % of total ajmalicine.     

长春花激素完全适应型细胞的生长和阿玛碱合成特性

从长春花激素依赖型细胞系（C20D）筛选出一种激素完全适应型的细胞系（C20hi),考察了两种细胞生长、阿玛碱合成和引吲哚生物碱生物合成相关的酶的活性,结果表明：在生长培养基上二生长无显差异,而C20hi细胞平均阿玛碱含量是C20D的31.9倍,在生产培养基上C20hi细胞生长较C20D快,C20hi平均阿玛碱含量是C20D的18.4倍。通过比较生产和生长培养基中C20hi细胞的色氨酸脱羧酶、异胡豆苷合酶和long牛儿醇-10-脱氢酶活性,发明,通过5年的继代培养,激素完全适应型细胞系C20hi的阿玛碱含量是比较稳定的。     

长春花激素完全适应型细胞的生长和阿玛碱合成特性

从长春花激素依赖型细胞系(C20D)筛选出一种激素完全适应型的细胞系(C20hi),考察了两种细胞生长、阿玛碱合成和与吲哚生物碱生物合成相关的酶的活性,结果表明:在生长培养基上二者生长无显著差异,而C20hi细胞平均阿玛碱含量是C20D的31.9倍,在生产培养基上C20hi细胞生长较C20D快,C20hi平均阿玛碱含量是C20D的18.4倍.通过比较生产和生长培养基中C20hi细胞的色氨酸脱羧酶、异胡豆苷合酶和牛儿醇-10-脱氢酶活性,发现在生产培养基中培养细胞的3种酶活性均显著高于生长培养基,但与阿玛碱积累没有密切关系.研究结果还表明,通过5年的继代培养,激素完全适应型细胞系C20hi的阿玛碱含量是比较稳定的.     

In situ adsorption for enhanced alkaloid production byCatharanthus roseus

Summary A significant fraction (10–40%) of the indole alkaloids produced byCatharanthus roseus was observed to be secreted into the medium. When a neutral polymeric resin, known to adsorb these alkaloids, was added to the cultivation medium, the accumulation of total indole alkaloids and the specific alkaloids, ajmalicine and serpentine were stimulated. Sorbent addition was also observed to result in increased ratios of ajmalicine to serpentine, which suggests the potential of using in situ adsorption to direct metabolism toward a specific product or intermediate within a given pathway.     

Studies on production of ajmalicine in shake flasks by multiple shoot cultures of Catharanthus roseus

The effects of different concentrations of indole-3-acetic acid (IAA) and benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and Skoog's (MS) medium with a high concentration of IAA (11.42 microM) and a low concentration of BA (2.22 microM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 microM) and a high concentration of BA (8.90 microM), shoots released high levels of ajmalicine into the culture medium. Quantification of ajmalicine was performed by high performance liquid chromatography (HPLC). The highest concentration of ajmalicine production (0.166% dry wt) was obtained by shoot cultures grown in MS medium containing IAA (11.42 microM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2-fold more in IAA (11.42 microM) supplemented medium compared with the high concentration of BA (8.90 microM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 microM) supplemented medium released the maximum production of ajmalicine (0.853 g/L) into the culture medium after 15 days of cultivation. The experimental data show that the secretion of ajmalicine was 2-fold more into the culture medium supplemented with a high concentration of BA compared to that with a low concentration of BA. Data presented here show that production of ajmalicine by shoot cultures is not correlated with growth rate. Dimeric indole alkaloids vincristine and vinblastine were not present in shoot cultures. Ajmalicine production by shoot cultures was 2.4-fold higher compared to leaves of 1-year-old naturally grown plants.     

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g^-1 DW, or 7.0 and 3.0 mg l^-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa methyl jasmonate - mU milliunits     

The effect of inoculum density and conditioned medium on the production of ajmalicine and catharanthine from immobilized Catharanthus roseus cells

The effect of the cell-inoculum size and the addition of conditioned medium on ajmalicine and catharanthine production were studied using immobilized Catharanthus roseus cells. Higher specific-uptake rates of ammonium, nitrate, and sugars were observed in the low-inoculum-density cultures (50 g FW/L) compared to the high-inoculum-density cultures (100 g FW/L). Alkaloid production was not correlated with the exhaustion of a particular nutrient from the medium. The high-inoculum-density cultures produced higher ajmalicine concentrations throughout the experiment. Catharanthine production was similar between the two inoculum-density cultures. The addition of conditioned medium to MS-production medium dramatically improved the production of ajmalicine and catharanthine. The addition of conditioned medium enhanced ajmalicine production from immobilized Catharanthus roseus cultures on day 15 by at least two- to fourfold compared to media without the conditioning factors. Catharanthine production was increased by nearly fivefold in cultures with conditioned medium compared to those without conditioned medium. The enhancing effects of conditioned medium on alkaloid production were attributed to an unidentified factor produced and secreted by suspension cultures of C. roseus. The presence of conditioned medium also decreased the sucrose hydrolysis rate. The ajmalicine concentration in these immobilized cell cultures was found to be a function of the fresh-weight concentration, irrespective of the inoculum density or the culture medium. The medium choice and the inoculum density determined how rapidly fresh weight was accumulated and thus, how quickly ajmalicine was produced. Ajmalicine production correlated positively with fresh-weight concentration, but catharanthine production was not correlated with fresh-weight concentration.     

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in moles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high-and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.Biotechnology Delft Leiden, Project Group Plant Cell Biotechnology Correspondence to: R. Verpoorte     

Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts

The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN₃ or 0.5 millimolar Cu²⁺. In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu²⁺, was insensitive to inhibition by NaN₃. The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide-insensitive component, which may be due to complexation with organic counterions and phenolics.     

Regulation of product synthesis in cell cultures of Catharanthus roseus. Effect of culture temperature

A three year old, alkaloid producing cell line of Catharanthus roseus, maintained at 25°C, was grown on 2% sucrose at various temperatures from 10° to 45°C. Growth rates were maximal at 35°C but declined rapidly above 35°C and below 25°C. Maximum serpentine yields reached a peak at between 20°C and 25°C and fell sharply above and below these temperatures, while ajmalicine showed a sharp peak of accumulation at 20°C. The variable serpentine/ajmalicine ratio at different growth temperatures suggests that lower temperatures may favour ajmalicine accumulation. Both the growth rate and the rate of alkaloid accumulation at 25°C were therefore sensitive to small changes in average culture temperature.     

Effects of stress factors, bioregulators, and synthetic precursors on indole alkaloid production in compact callus clusters cultures of Catharanthus roseus

Compact callus cluster (CCC) cultures established from Catharanthus roseus consist of cohesive callus aggregates displaying certain levels of cellular or tissue differentiation. CCC cultures synthesize about two-fold more indole alkaloids than normal dispersed-cell cultures. Our studies here show that additions of KCl, mannitol, and a variety of synthetic precursors and bioregulators to the CCC cultures markedly improved indole alkaloid production and release of these alkaloids into the medium. Treatment with 250 mM mannitol and 4 g/l KCl yielded 42.3 mg l(-1) and 33.6 mg l(-1)of ajmalicine, respectively; these amounts were about four-fold higher than the control. Succinic acid, tryptamine, and tryptophan feedings also significantly increased ajmalicine (41.5 mg l(-1), 36.9 mg l(-1), and 31.8 mg l(-1), respectively) and catharanthine (21.1 mg l(-1), 17.2 mg l(-1), and 18 mg l(-1), respectively) production by the CCC cultures, while geraniol feeding inhibited biomass and alkaloid accumulation. We also found that tetramethyl ammonium bromide could significantly improve ajmalicine production (49.3 mg l(-1)) and catharanthine production (18.3 mg l(-1)) in C. roseus CCC cultures. The mechanisms responsible for these treatment effects are discussed herein.     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

Two-stage batch process for the production of ajmalicine by Catharanthus roseus: The link between growth and production stage

The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. (c) 1995 John Wiley & Sons, Inc.     

Economic assessment of plant cell culture for the production of ajmalicine

Cost estimates have been prepared for commercial-scale production of ajmalicine-rich Catnaranthus roseus biomass using plant cell culture. At the current state of the technology the cost would be approximately $7.30/lb dry biomass ($3215/kg ajmalicine). Naturally-grown C. roseus roots have a 50% lower ajmalicine concentration but would cost only ca. $0.70/lb ($619/kg ajmalicine). The principal reason for the high cost of the plant cell culture route is not the slow specific growth rate (0.35 day(-1)), but rather the slow specific product accumulation rate (0.26 mg/g day). This rate will have to be increased by a factor of 40 to make the process competitive.     

Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don. and its conversion into serpentine

Isolated vacuoles from ajmalicine-producing cell suspensions of Catharanthus roseus accumulated the alkaloid ajmalicine. Dissipation of the transtonoplast pH gradient with nigericin abolished ajmalicine accumulation, whereas dissipation of the transtonoplast potential with valinomycin had no effect. Addition of Mg-ATP resulted in a higher ajmalicine accumulation. Serpentine produced by the cells was largely recovered in isolated vacuoles; in contrast, ajmalicine was lost. Ajmalicine was converted in vitro into serpentine by horseradish basic peroxidases (EC 1.11.1.7). In cultured cells there was a striking conformity between the time course of serpentine content and that of the activity of basic peroxidases. Ajmalicine was converted efficiently into serpentine by basic peroxidases extracted from vacuoles and by intact isolated vacuoles. The results are consistent with the hypothesis that ajmalicine accumulates by an ion-trap mechanism and that the accumulated ajmalicine is converted into serpentine inside the vacuoles. By the transformation of ajmalicine into the charged serpentine a trap is created to retain the alkaloids more efficiently in the vacuole.Abbreviations and Symbols DCCD N,N-dicyclohexylcar-bodiimide - TPP⁺ tetraphenylphosphonium - pH trans-tonoplast pH gradient - transmembrane potential difference We thank Dr W. Kreis, Universität Tübingen, FRG for fruitful discussions and for his suggestions in isolation of vacuoles.     

The effect of ajmalicine spiking and resin addition timing on the production of indole alkaloids from Catharanthus roseus cell cultures

The potential for the feedback inhibition of indole alkaloid synthesis was investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or 18 mg/L ajmalicine on day 0. The production of ajmalicine, catharanthine, and serpentine were inhibited in a dose-dependent manner. The inhibition was transient as the exogenous ajmalicine was ultimately either metabolized in the medium or within the cell. The addition of neutral resin has previously been shown to enhance ajmalicine production. To minimize product inhibition and product metabolism, Amberlite XAD-7 resin was added to immobilized cultures of C. roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for spent resin every 5 days. The addition of resin did not decrease the viability of the culture. Growth was reduced only in cultures with resin added on day 0. Alkaloid production was enhanced to different extents by the timing of resin addition, suggesting that feedback inhibition or product metabolism was present throughout the culture period. Ajmalicine recovery was nearly 100% when the resin was added initially either on day 0 or day 5. Ajmalicine recovery was reduced to 55% when the resin was added later in the culture period starting on day 15, presumably because of resin saturation or the inaccessibility of alkaloids trapped in the vacuole. Delaying the addition of XAD-7 resin until 5 days after the start of the culture resulted in the highest improvement in ajmalicine production, i.e approximately 70% and also resulted in the complete recovery of ajmalicine from the cell.