Alkaloid Production Medium Research Articles

Structure and Synthesis of a New Indole Alkaloid, 19 (S)-Hydroxy- Nb-methylraumacline, Obtained by the Biotransformation of Ajmaline in Plant Cell Cultures of Rauwolfia Serpentina Benth.

From the plant cell suspension cultures of Tauwolfia serpentina Benth., which were cultivated in the alkaloid-production medium after feeding of ajmaline (1), a new indole alkaloid 19- ( S)-hydroxy- N b-methylraumacline ( 4) was isolated. The structure of 4 first elucidated by spectroscopic analysis was determined by the chemical synthesis from ajmaline ( 1).

Effect of culture process on alkaloid production by Catharanthus roseus cells : I. Suspension cultures

The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenk's alkaloid production medium (APM) were evaluated. The 1-stage process consisted of inoculating APM and incubating for 15 days. The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM. Growth, main nutrient consumption and alkaloid production were monitored. Both culture processes produced ∼ 20 g dw per 1 of biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (> 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg l −1 and 25 to 4000 μg per g dw, respectively) for 2-stage than for 1-stage cultures. For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in production. Small but significant (∼ 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.

Effect of culture process on alkaloid production by Catharanthus roseus cells : II. Immobilized cultures

Two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of Catharanthus roseus cells using Zenk's Alkaloid Production Medium (APM) were evaluated. The 1-stage process consisted of inoculating APM containing bioreactors and incubating for 15 d. The 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with APM. The production stage which lasted for 15 d. High production in 2-stage cultures required the replacement of the growth regulator 2,4-dichlorophenoxyacetic acid by indole-3-acetic acid in the growth medium and a growth stage of 6 d (late exponential phase) before production initiation. Growth, main nutrient consumption and alkaloid production were monitored. Both culture regimes resulted in similar biomass production, dw (10–13 g l −1). The 2-stage cultures yielded biomass richer in organic nutrients (200–300%) and with higher respiratory activity (∼ 250%), indicated by their lower biomass-to-carbohydrate yields (31% and 26%), as compared to 1-stage cultures (41%). Two-stage cultures produced more known products (10 as compared to 6) at yields (5 to 4800 μg g −1) 3 to 5 times higher than 1-stage cultures. More alkaloids were alkaloids released in the medium of 2-stage cultures, under non-lysing conditions, (20 to 4700 μg l −1) than in 1-stage cultures (20 to 460 μg l −1). These results were compared to those obtained from shake flask cultures performed at the same time, with the same C. roseus cell line and under similar regimes and reported previously. Suspension and immobilized cultures performed according to the 1-stage regime showed similar total production. However, release of known alkaloids was 2 to 3 times higher in immobilized than in suspension cultures. Total alkaloid production of 2-stage suspension cultures was 3.8-fold higher than 2-stage immobilized cultures. Two stage immobilized cultures released 4 more known alkaloids than the 2-stage suspensions. Lower oxygen availability in the 2 l immobilized cultures may explain lower specific growth rates (0.15–0.22 d −1) and total alkaloid production levels, compared to 200 ml suspension cultures (0.2–0.4 d −1) reported in our previous paper.

Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies

Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 μg l −1), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (< 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increases in total alkaloid (from ∼ 5–10 mg l −1 to 15 mg l −1), ajmalicine (from 0 to ∼ 0.29 mg l −1) and serpentine (from 0 to ∼ 0.20 mg l −1) release, without apparent cell lysis or decrease in the culture viability. This product release was estimated to represent 100% of alkaloids produced.

Mass Spectral Evidence of the Occurrence of Vindoline in Heterotrophic Cultures ofCatharanthus roseusCells

Vindoline concentrations in the leaves of 70 CATHARANTHUS ROSEUS of 3 cultivars were analyzed by HPLC, and 3 plants were selected for starting callus cultures on different media. When the initial calli were analyzed using a vindoline-specific RIA, the assay suggested a vindoline content of about 10 (-5)% dry weight for one-third of the first 60 cultures examined. Due to the unexpectedly high incidence of vindoline-positive calli, the screening programme was discontinued and efforts were concentrated on verifying the existence of this alkaloid in the cells. Suspension cultures derived from the 5 most immunopositive calli in an alkaloid production medium were analyzed by HPLC and GC/MS. Comparison with reference material showed that the heterotrophic suspension cultures contained a compound identical to vindoline.

Improved production of raucaffricine by cultivated Rauwolfia cells

The production of the glucoalkaloid raucaffricine has been optimized in cell suspension cultures of Rauwolfia serpentina. In an alkaloid production medium modified by adding 100 g sucrose and 2.5 9 MgSO 4·.7H 2O/l of medium, the culture produced after 18 days up to 1.6 g raucaffricine when 11 medium was inoculated with 200 g of cells grown for 10 days in Linsmaier-Skoog medium. This yield exceeds that known from intact plants ( Rauwolfia caffra) by a factor of 12 and is the fifth highest value for a secondary plant metabolite produced by cultivated plant cells.

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.

Characterization of a novel N-methyltransferase (NMT) from Catharanthus roseus plants

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615-767 and 916 could not be induced to produce DAT or NMT enzyme activities.

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.

Alkaloid formation by habituated and tumorous cell suspension cultures of Catharanthus roseus

Habituated and tumorous Catharanthus roseus cells grown in the absence of hormones accumulated indole alkaloids. Total alkaloids and alkaloid pattern were the same when cells were cultured in medium without hormones or in alkaloid production medium with and without indole acetic acid. Treatment of cells with Pythium homogenate as elicitor did not increase total alkaloids or change the pattern of alkaloids produced. When either habituated or tumorous cells were grown in 1B5 medium after Gamborg et al (1968) containing 2,4-dichlorophenoxyacetic acid (2,4-D), their capacity to accumulate alkaloids decreased with time. The levels of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) specific activities were constant throughout growth except when cells were exposed to 2,4-D in 1B5 medium, where enzyme activities declined in step with the decrease in alkaloid accumulation. Neither habituated nor tumorous cell suspension cultures accumulated vindoline, nor could they be induced to produce this alkaloid by any of the given treatments.

Ultrastructure ofCatharanthus roseus cells culturedin vitro and exposed to conditions for alkaloid accumulation

Periwinkle (Catharanthus roseus) cells cultured in 1-B 5 medium display the ultrastructure of parenchyma cells. The parenchyma character remained unchanged when cells were exposed to any one of three different conditions effecting alkaloid accumulation. Transfer of cells to “alkaloid production medium” for 2 weeks (condition 1) accorded two special features,i.e., unusually big lipid droplets in the cytoplasm and, upon fixation, one or several electron-dense droplets of spongy precipitate in vacuoles. Among hormone-autotrophic cultures (condition 2) some cells showed a fine electron-dense vacuolar precipitate. Addition ofPhythium homogenate (fungal elicitor) to cells cultured in 1-B 5-medium for 10 days (condition 3), cells showed a frequent appearance of singular big lipid droplets in the cytoplasm, whereas vacuoles remained devoid of precipitate. The appearance of big lipid droplets and of vacuolar precipitate is interpreted as progressing cytodifferentiation, but is coincidental with alkaloid accumulation.

Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus

Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.

Greening and strictosidine lactam production in two cell lines of Vinca major cv. variegata

Two cell lines of Vinca major L. cv. variegata produced strictosidine lactam as the main alkaloid. Quantities varied from 500–1000 μg/g DW in the high-yielding line and 1–100 μg/g DW in the low-yielding line. Transfer of cells to alkaloid production medium resulted in a 6–8 fold increase in alkaloid production with the high-yielding line, some increase in the low-yielding line and in both lines induced intense greening (up to 200 μg chlorophyll/g DW) indicating chloroplast differentiation. Though in the source plant leaves are the main storage site, no correlation between alkaloid accumulation and chloroplast differentiation could be found.

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.

Influence of production media on Cinchona cell cultures; spontaneous formation of β-carbolines from L-tryptophan

Evidence is presented that the β-carboline alkaloids norharman and harman are artifacts formed from L-tryptophan. Transfer of Cinchona ledgeriana suspension cultured cells to Zenk's alkaloid production medium (ZAP medium) resulted in cell death. No quinoline or terpenoid indole alkaloids were formed. However, the L-tryptophan present in the production medium was transformed to the β-carboline alkaloids, harman and norharman. It was demonstrated that norharman was also formed in ZAP medium without cells and in ZAP medium containing frost-killed cell material.

Regulation of product synthesis in cell cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle Catharanthus roseus (L) G. Don were maintained on Gamborg's B5 medium and their growth monitored by measuring cellular fresh and dry weight, cell number and mitotic activity. Samples of cells of different ages and physiological states were subcultured onto an alkaloid production medium and their rates of growth and alkaloid accumulation measured over a period of 30–45 days. In two experiments the rate of biomass accumulation was directly related to the rate of cellular serpentine accumulation. Possible mechanisms underlying this phenomenon are discussed in relation to the properties of cells comprising the inocula.

RLCC-isolation of Raucaffricine from its most efficient source ? cell suspension cultures of Rauwolfia serpentina Benth

Raucaffricine (vomilenine-galactoside) was shown to be the major indole alkaloid of Rauwolfia serpentina Benth. cell suspension cultures grown in AP-medium (alkaloid production medium). Several grams of this glycoalkaloid can conveniently be isolated by RLCC (Rotation Locular Countercurrent Chromatography). A newly discovered enzyme efficiently converts the glycoalkaloid to its aglycon, vomilenine, which occupies a key function in the biosynthesis of ajmaline. This is the first demonstration of the occurrence of raucaffricine in Rauwolfia serpentina.

Alkaloid production in Catharanthus roseus cell cultures: Initial studies on cell lines and their alkaloid content

Several hundred serially cultured cell suspensions derived from three cultivars of periwinkle ( Catharanthus roseus) were established in Gamborg's B5 medium and then transferred to Zenk's alkaloid production medium. Total alkaloid concentration ranged from 0.1 to 1.5% of dry weight. Alkaloids present were of the corynanthe, strychnos and aspidosperma types, with the greatest diversity arising during the third to the fifth week of subculturing. The alkaloid content appeared both specific for, and reproducible in, individual cell lines.