Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus

The effects of terpenoid precursor feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid     

Insulin regulation of amino acid transport in mesenchymal cells from avian and mammalian tissues.

Insulin regulation of amino acid transport across the cell membrane was studied in a variety of mesenchymal cell directly isolated from avian and mammalian tissues or collected from confluent cultures. Transport activity of the principal systems of mediation in the presence and absence of insulin was evaluated by measuring the uptake of representative amino acids under conditions approaching initial entry rates. Insulin enhanced the transport rate of substrate amino acids from the A system(alpha-aminoisobutyric acid, L-proline, glycine, L-alanine and L-serine) in fibroblasts and osteoblasts from chick-embryo tissues, in mesenchymal cells (fibroblasts and smooth muscle cells) from immature rat uterus, in thymic lymphocytes from young rats and in chick-embryo fibroblasts from confluent secondary cultures. In these tissues, the uptake of amino acid substrates of transport systems L and Ly+ (L-leucine, L-phenylalanine, L-lysine) was not affected by the presence of the hormone. No insulin control of amino acid transport was detected in chick-embryo chondroblasts and rat peritoneal macrophages. These observations identify the occurrence of hormonal regulatory patterns of amino acid transport for different mesenchymal cells types and indicate that these properties emerge early during cell differentiation.     

Some characteristics of tryptophan uptake in Claviceps species

Tryptophan serves as a precursor for the biosynthesis of alkaloids in the ergot fungus, Claviceps purpurea (Fries) Tulasne, and also is believed to act as an inducer of the enzymes necessary for alkaloid production. The characteristics of the transport system responsible for the accumulation of tryptophan in ergot mycelium were investigated, with the goal of clarifying the complex relationships among tryptophan uptake, size of the free intracellular pool of tryptophan, and alkaloid production. The characteristics of tryptophan uptake were studied by pulse feeding radioactively labeled tryptophan to cultures of Claviceps species, strain SD-58, which represented a variety of ages and nutritional states. Tryptophan accumulation in strain SD-58 is mediated by an energy-requiring system which exhibits specificity for neutral aromatic and aliphatic l-amino acids, is pH and temperature dependent, and shows saturation at high substrate concentrations. Tryptophan transport is a function of the intracellular concentration of free tryptophan, the nitrogen deficiency of the mycelium, the rate of growth, and alkaloid production, which were measured in Claviceps strain SD-58 growth in several culture media, some of which promoted alkaloid production and some of which did not. The results indicate that the initial velocity of tryptophan transport is not directly related to alkaloid production.     

Transgenic periwinkle tissues overproducing cytokinins do not accumulate enhanced levels of indole alkaloids

Cytokinins play a critical role in several aspects of plant growth, metabolism and development. We previously reported that adding cytokinins to the culture medium of a suspension-cultured cell line of periwinkle increased the accumulation of indole alkaloids, and our aim was to compare the effect of exogenously-applied cytokinins with that of elevated levels of endogenous cytokinins on indole alkaloid production. We used an Agrobacterium tumefaciens strain yielding a plasmid with the isopentenyl transferase gene under control of its own promoter. Co-culture of suspension cells with the bacteria caused a severe stress response leading to cell necrosis. Therefore, we failed to transform this material but we succeeded in transforming periwinkle cotyledons. We verified that callus cultures generated from the isopentenyl transferase-transgenic cotyledons accumulated high cytokinin concentrations. Treating normal callus cultures (generated from untransformed cotyledons) with cytokinins enhanced their alkaloid production. By contrast, the enhanced concentration of endogenous cytokinins in transgenic calli did not increase indole alkaloid production, and thus did not mimic the effect of exogenously-applied cytokinins. Hypothesis to explain this discrepancy are discussed.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - DW dry cell mass - ipt isopentenyl transferase gene     

Stimulation of berberine secretion and growth in cell cultures of Thalictrum minus

Summary An endo-pectate lyase (PL; EC 4.2.2.2), originally cloned fiom the phytopathogenic bacterium Erwinia chrysanthemi EC16, was expressed in recA ^– E. coli strain DK1, purified to a single band by isoelectric focusing and used to induce berberine production in established plant suspension cultures of Thalictrum minus L. subsp. saxatile. Addition of 10^–9M pectate lyase c (PLc) stimulated berberine production and enhanced secretion of the alkaloid into the medium. A lower concentration of PLc, 10^–11M, stimulated a transient two-fold increase in cell growth rate relative to untreated cultures. Parallel changes in L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity with the rate of berberine synthesis and the inverse relationship between cell growth and berberine synthesis imply that berberine synthesis is stress-related in this cell line.     

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.     

Effect of ethylene on sanguinarine production from Papaver somniferum cell cultures

A Papaver somniferum cell line capable of producing sanguinarine equivalent to 3% of cell dry weight was used to determine if ethylene was involved in signalling the biosynthesis of this alkaloid. A 3.3-fold increase in ethylene emanation from these cell suspension cultures was observed 7 h after elicitation with a Botrytis fungal homogenate. The rate of ethylene release then decreased to near zero after 48 h, suggesting that a pulse of ethylene production may be involved in sanguinarine production. However, sanguinarine biosynthesis was not promoted when either the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), or the ethylene releasing agent, 2-chloroethylphosphonic acid (ethephon), was added to the culture. These results strongly suggest that ethylene is not intimately involved in the production of sanguinarine from Papaver somniferum cell cultures or in the transduction of the elicitation event.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid     

Uncharacteristic alkaloid synthesis by suspension cultures of Cinchona pubescens fed with L-tryptophan

The growth and alkaloid production of a liquid suspension culture of Cinchona pubescens has been studied, particularly with attention to the effect on the alkaloid spectrum of feeding cultures with L-tryptophan. This treatment did not enhance the production of any of the known alkaloids of Cinchona. Above 2mmM, however, the presence of the amino acid was toxic, causing extreme acidification of the medium and cell death. Under these conditions a number of indole and quinoline derivatives accumulated. The principal component of the alkaloid fraction proved to be norharman; indole-3-aldehyde was also isolated. Both these products probably occur by uncharacteristic metabolism of L-tryptophan. Furthermore, evidence for the degradation of endogenous alkaloids was obtained, as 4-hydroxymethylquinoline was also isolated. None of the known quinoline alkaloids of Cinchona, which were present in untreated cells, could be detected after L-tryptophan treatment, even when large amounts of culture were analysed. It is concluded that, in this instance, Cinchona alkaloid production cannot be improved by feeding with a precursor.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BA benzyladenine     

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor-and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor-and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.     

Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Effect of tweens on the production of ergot alkaloids byAspergillus fumigatus

Tween 80, which caused increased biomass formation, also produced the highest increase in the uptake rate of all components of the medium. The fatty acid components of the respective Tweens,i.e. palmitic acid (Tween 40), stearic acid (Tween 60), and oleic acid (Tween 80), have no effect either on alkaloid production or on substrate uptake. The fatty acid composition was different in the cell membrane of the culture supplemented with Tween 60 and facilitated the transport of metabolites into the cells.     

Medium- and light-induced formation of serpentine and anthocyanins in cell suspension cultures of Catharanthus roseus

By irradiation with fluorescent light, medium-induced cell cultures of Catharanthus roseus accumulated anthocyanins and the indole alkaloid serpentine. The formation of both compounds was inhibited by phosphate and nitrogen-containing mineral salts and stimulated by high sucrose concentrations. The accumulation of serpentine was preceded by an increase and subsequent decrease of its biogenetic precursor ajmalicine, which was the predominant alkaloid of medium-induced cultures in the dark. High concentrations of serpentine or anthocyanins were observed only in a small proportion of all cells present in a medium-induced culture. The aglycones of the anthocyanins were identified as petunidin, malvidin and hirsutidin.     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.     

pH-dependent uptake of fumaric acid in Saccharomyces cerevisiae under anaerobic conditions

Microbial production of C(4) dicarboxylic acids from renewable resources has gained renewed interest. The yeast Saccharomyces cerevisiae is known as a robust microorganism and is able to grow at low pH, which makes it a suitable candidate for biological production of organic acids. However, a successful metabolic engineering approach for overproduction of organic acids requires an incorporation of a proper exporter to increase the productivity. Moreover, low-pH fermentations, which are desirable for facilitating the downstream processing, may cause back diffusion of the undissociated acid into the cells with simultaneous active export, thereby creating an ATP-dissipating futile cycle. In this work, we have studied the uptake of fumaric acid in S. cerevisiae in carbon-limited chemostat cultures under anaerobic conditions. The effect of the presence of fumaric acid at different pH values (3 to 5) has been investigated in order to obtain more knowledge about possible uptake mechanisms. The experimental results showed that at a cultivation pH of 5.0 and an external fumaric acid concentration of approximately 0.8 mmol · liter(-1), the fumaric acid uptake rate was unexpectedly high and could not be explained by diffusion of the undissociated form across the plasma membrane alone. This could indicate the presence of protein-mediated import. At decreasing pH levels, the fumaric acid uptake rate was found to increase asymptotically to a maximum level. Although this observation is in accordance with protein-mediated import, the presence of a metabolic bottleneck for fumaric acid conversion under anaerobic conditions could not be excluded.     

Production of higher levels of trigonelline by cell cultures of Trigonella foenum-graecum than by the differentiated plant

Callus cultures of Trigonella foenum-graecum contained 3 to 4 times more trigonelline than the seeds of this plant and 12 to 13 times more than the roots and shoots. Even higher levels of this alkaloid were produced by suspension cultures. This high productivity was maintained during successive subculturing of calli and cell suspensions for eight months. Thus, trigonelline is to be added to the group of the few metabolites whose synthesis in cell cultures exceeds its production in the differentiated plants. Media that had supported the growth of suspension cultures contained one third or more of the total alkaloid, whereas media of callus cultures contained about one tenth of this substance. Trigonelline accumulated in callus and suspension cultures with aging. Raising the level of nicotinic acid in the nutrient medium resulted in some increase of trigonelline production by the culture.Abbreviations 2.4 D 2.4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IPA indolepropionic acid - NAA -naphthaleneacetic acid - GA Gibberellic acid - K kinetin     

Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress

Uric acid is considered a major antioxidant in human blood that may protect against aging and oxidative stress. Despite its proposed protective properties, elevated levels of uric acid are commonly associated with increased risk for cardiovascular disease and mortality. Furthermore, recent experimental studies suggest that uric acid may have a causal role in hypertension and metabolic syndrome. All these conditions are thought to be mediated by oxidative stress. In this study we demonstrate that differentiation of cultured mouse adipocytes is associated with increased production of reactive oxygen species (ROS) and uptake of uric acid. Soluble uric acid stimulated an increase in NADPH oxidase activity and ROS production in mature adipocytes but not in preadipocytes. The stimulation of NADPH oxidase-dependent ROS by uric acid resulted in activation of MAP kinases p38 and ERK1/2, a decrease in nitric oxide bioavailability, and an increase in protein nitrosylation and lipid oxidation. Collectively, our results suggest that hyperuricemia induces redox-dependent signaling and oxidative stress in adipocytes. Since oxidative stress in the adipose tissue has recently been recognized as a major cause of insulin resistance and cardiovascular disease, hyperuricemia-induced alterations in oxidative homeostasis in the adipose tissue might play an important role in these derangements.     

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 approximately 20 g dw per 1 biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (greater than 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg l-1 and 25 to 4000 micrograms 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 (approximately 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.