Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering

Catharanthus roseus plants produce many pharmaceutically important indole alkaloids, of which the bisindole alkaloids vinblastine and vincristine are antineoplastic medicines and the monoindole alkaloids ajmalicine and serpentine are antihypertension drugs. C. roseus cell cultures have been studied for producing these medicines or precursors catharanthine and vindoline for almost four decades but so far without a commercially successful process due to biological and technological limitations. The research thus focused on the one hand on engineering the bioreactor process on the other engineering the cell factory itself. This review mainly summarizes the progress made on biochemical engineering aspects of C. roseus cell cultures in bioreactors in the past decades and metabolic engineering of indole alkaloid production in recent years. The paper also attempts to highlight new strategies and technologies to improve alkaloid production and bioreactor performance. Perspectives of metabolic engineering to create new cell lines for large-scale production of indole alkaloids in bioreactors and effective combination of these up- and down-stream processing are presented.     

Recent Advances in Indole Alkaloid Production by Catharanthus roseus (Periwinkle)

Considerable progress has been accomplished in cell culturetechniques for production of indole alkaloids fromCatharanthusroseus. This paper reviews the advances made in this area. Keyeffects of nutrients, environmental effects, stress-inducingcompounds and strain selection techniques on the productionof alkaloids are summarized. Cultivation methods such as suspensioncultures, immobilization, and a novel biofilm configurationare compared. Economic considerations in the large scale productionof secondary metabolites are covered. Indole alkaloids, cellculture techniques, Catharanthus roseus. Key words: Indole alkaloids, cell culture techniques, Catharanthus roseus     

Effects of Inorganic Phosphate on the Utilization of Sucrose by Suspension-cultured Catharanthus roseus Cells

The metabolic fate of [U-¹⁴C]sucrose in suspension culturesof Catharanthus roseus cells was monitored for 96 h after thecells were transferred to fresh complete (‘+Pi’)or to phosphate-deficient Murashige and Skoog (‘–Pi’)medium. Sucrose was hydrolysed extracellularly to glucose andfructose. The rate of uptake of sugars by the cells was 1.5–3times higher in ‘+Pi’ culture than in ‘–Pi’culture. Little difference in the rate of incorporation of radioactivityinto the ethanol-soluble fraction was found between the ‘+Pi’and ‘– Pi’ cultures during the initial 24h of culture, but after 48 h the rate in ‘ +Pi’cultures was higher than that in ‘–Pi’ cultures.Incorporation of radioactivity into ethanol-insoluble macromoleculeswas always significantly higher in the cells in ‘+Pi’cultures than in those in ‘–Pi’ cultures.The results suggest that Pi strongly affects the utilizationof sugars by cultured plant cells through the stimulation oftransport of sugars as well as through the activation of metabolism. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, sucrose, transport, metabolism     

Effect of Spermidine and Methylglyoxal-bis (Guanyl-hydrazone) (MGBG) on In Vitro Pollen Germination and Tube Growth in Catharanthus roseus

Incorporation of polyamine-spermidine into the nutrient mediumat 10^–6 and 10^–5 M concentrations stimulates pollen-tubegrowth in vitro in Catharanthus roseus L. G. Don. MGBG, an inhibitorof spermidine biosynthesis, at 0.5 x 10^–3 and 1 x 10^–3M concentrations reduced the percentage of germination as wellas tube growth and at a concentration of 1.5 x 10^–3 Mgermination was totally inhibited. Pollen grains incubated inthe medium containing 1.5 x 10^–3 M MGBG, when transferredto a fresh medium with 10^–5 M spermidine, resulted in80% germination recovery, along with considerable tube growth.Experiments with actinomycin-D indicate that stimulation ofpollen-tube growth by spermidine may involve de novo synthesisof protein. Catharanthus roseus, pollen germination, tube growth, spermidine, MGBG, inhibition, actinomycin-D     

Role of elements and physiologically active compounds in the regulation of synthesis and accumulation of indole alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L.

Effects of various elements (Co, Ni, Zn, W, Mn, Cr, B, Mo, Fe, and V), natural and synthetic auxins, cytokinins, and gibberellin on biosynthesis and accumulation of indole alkaloids was studied at increasing concentrations in the model system of Madagascar periwinkle seedlings (Catharanthus roseus L.). The main types of concentration dependences for the effect of physiologically active compounds under study were evaluated. A possible mechanism of the influence of Zn and auxin on this process was partly clarified. The compounds were shown to modulate various stages in the biosynthesis of monomeric indole alkaloids (catharanthine and vindoline).__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 340–346.Original Russian Text Copyright © 2005 by Lovkova, G. Buzuk, Sokolova, L. Buzuk.     

Salt Tolerance in the Triticeae: Growth and Solute Accumulation in Leaves of Thinopyrum bessarabicum

Gorham, J., McDonnell, E., Budrewicz, E. and Wyn Jones, R. G.1985. Salt tolerance in the Triticeae: growth and solute accumulationin leaves of Thinopyrum bessarabicum.—J. exp. Bot. 36:1021–1031. The diploid wheatgrass Thinopyrum bessarabicum was found towithstand prolonged exposure to 350 mol m^–3 NaCl in hydroponicculture. During the gradual addition of salt to the externalmedium, osmotic adjustment was rapidly achieved by the accumulationof Na and Cl. Following osmotic adjustment constant leaf Naand Cl concentrations were maintained, and K was retained ata high level. Thinopyrum bessarabicum may be described as anosmoconformer, adjusting its internal osmotic pressure to 400–500mOsmol kg^–1 above that of the external medium in hydroponicculture. Both slower shoot initiation and reduced leaf lengthcontributed to the reduced growth rates at higher salinities.Leaf width was not affected. Increasing salinity resulted inincreases in leaf concentrations of phosphate, glycinebetaine,sucrose and proline, and in decreases in the concentrationsof nitrate, sulphate, magnesium, calcium, total amino acidsand organic acids. Thinopyrum bessarabicum exhibits salt tolerancecharacters which may be useful in wheat breeding. Key words: Salt stress, solute accumulation, osmotic adjustment, Thinopyrum     

Elicitor-mediated induction of phenylalanine ammonia lyase and tryptophan decarboxylase: Accumulation of phenols and indole alkaloids in cell suspension cultures of Catharanthus roseus

Cell suspension cultures (cell line No 615) of Catharanthus roseus cv. Little Delicata responded to elicitor treatment by accumulating monoterpenoid indole alkaloids and phenolic compounds. The excretion of phenols into the culture medium resulted from the induction of the branch-point enzyme phenylalanine ammonia lyase. The accumulation of alkaloids, however, occurred several hours earlier than the elicitor-mediated induction of tryptophan decarboxylase through which shikimate pathway intermediates are channelled into tryptamine and related indole alkaloids. The results indicate that both pathways for phenol and indole alkaloid biosynthesis responded to elicitor treatment and that no obvious causal relationship between pathways could be deduced from this study.Abbreviations PAL phenylalanine ammonia lyase - TDC tryptophan decarboxylase Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

Compact callus cluster suspension cultures of Catharanthus roseus with enhanced indole alkaloid biosynthesis

Summary Compact callus clusters showing a certain level of cellular or tissue differentiation were established from Catharanthus roseus stem and leaf explants in a modified MS liquid induction medium supplemented with 5.37 μM α-naphthaleneacetic acid and 4.65 μM kinetin. In the induction medium most leaf explants developed into friable half-closed hollow callus clusters, whereas in the same medium containing 2,4-dichlorophenoxyacetic acid instead of α-naphthaleneacetic acid, most leaf explants were induced to form dispersed cell suspension cultures. Characteristics of these different types of suspension cultures were compared, and the results showed that the compact callus clusters could synthesize indole alkaloids 1.9- and 2.4-fold higher than the half-closed hollow callus clusters and dispersed cell cultures, respectively. The degree of compaction expressed by the ratio of fresh weight to dry weight of these suspension cultures was correlated to indole alkaloid production. Our studies also postulated that the level of cellular/tissue differentiation might be responsible for these different alkaloid synthesis capabilities. Sucrose regime affected some properties (the size, degree of compaction, differentiation level) of the compact callus cluster cultures and therefore influenced alkaloid production.     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

Intercellular and Intercultural Heterogeneity in Secondary Metabolite Accumulation in Cultures of Catharanthus roseus following Cell Line Selection

Hall, R. D. and Yeoman, M. M. 1987. Intercellular and interculturalheterogeneity in secondary metabolite accumulation in culturesof Catharanthus roseus following cell line selection.—J.exp. Bot. 38: 1391–1398. Anthocyanin accumulation in a stock culture of Catharanthusroseus was consistently found, using microscopic and microdensitometrictechniques, to involve only c. 10% of the cell population. However,an analysis of 26 cell lines isolated from this culture hasindicated that all of the cells within the culture were, theoretically,capable of anthocyanin synthesis. Nevertheless, these linesdid display substantially different capacities for anthocyaninaccumulation. Detailed studies on individual cells from thesecultures have revealed that the variation in accumulation wasprimarily due to differing proportions of pigmented (i.e. productive)cells rather than differing mean intracellular anthocyanin concentrationswithin these cells. Both the proportion of productive cellsand the overall culture yield of the cell lines varied by >30-fold whereas the mean intracellular anthocyanin concentrationvaried by < 2-fold. The relevance of these results to thepossible control mechanisms involved in secondary metaboliteproduction in this and other culture systems is discussed. Key words: Catharanthus roseus, cell culture, anthocyanin, heterogeneity     

Production of Potato Spindle Tuber Viroid (PSTV) by Large Scale Fermentation of PSTY-Infected Potato Cell Suspension Cultures

Berlin, J., Wray, V., Forche, E., Reng, H.–G , Schler,H, Luckinger, R. and Mhlbach, H.–P. 1985. Production ofpotato spindle tuber viroid (PSTV) by large scale fermentationof PSTV–infected potato cell suspension cultures.—J.exp. Bot 36: 1985–1995. Cell suspension cultures of Solatiumdemissum, infected with the potato spindle tuber viroid (PSTV),were scaled up to volumes of up to 800 dm³ to provide sufficientand uniform plant material for subsequent studies on viroidbiosynthesis. Here we describe the technological aspects ofproducing the required amounts of biomass and viroid. The cells,which had been maintained on a medium containing expensive coconutmilk, were first adapted to rapid growth on the less expensiveB5–medium. The physiological state of the cells was monitoredby in vivo ³¹P–NMR spectroscopy Under the chosen conditionsthe scale–up from 10 dm³ inoculum from shake flasks tothe harvest of the 800 dm³ stirred fermenter lasted 38 d andprovided 112 kg biomass. Growth characteristics and viroid productionin shake flasks and large bioreactors were rather similar. Gelelectrophoretic analysis of isolated nucleic acids using silverstaining and Northern blot hybridization revealed a PSTV–contentof approximately 700 µg PSTV per kg fresh mass of culturedcells. Key words: Solanum demissum, plant cell cultures, potato spindle tuber viroid, biomass production, fermentation, in vivo ³¹P-NMR     

Inhibition of Somatic Embryogenesis in Tissue 2Medicago sativa by Aminoethoxyvinylglycine, Amino-oxyacetic acid, 2, 4-Dinitrophenol and Salicylic Acid at Concentrations which do not Inhibit Ethylene Biosynthesis and Growth

Meijer, E. G. M. and Brown, D. C. W. 1988. Inhibition of somaticembryogenesis in tissue cultures of Medicago sativa by aminoethoxyvinylglycine,amino-oxyacetic acid, 2, 4-dinitrophenol and salicylic acidat concentrations which do not inhibit ethylene biosynthesisand growth. J. exp. Bot. 39: 263–270. The effects of aminoethoxyvinyglycine (AVG), amino-oxyaceticacid (AOA), 2, 4-dinitrophenol (DNP) and salicylic acid (SA)on ethylene production, tissue proliferation and somatic embryo-genesisin a recently developed rapid in vitro regeneration system ofMedicago sativa L. were examined. Contrary to numerous publications,AVG, AOA and DNP did not affect the rate of ethylene biosynthesis,while SA even caused an increase in ethylene production. Allfour compounds were, however, potent inhibitors of somatic embryoformation in the M. sativa tissue cultures, even at concentrationswhich did not affect tissue growth. Generally, a 5-d exposureto the inhibitors reduced the number and quality of somaticembryos obtained. It is suggested that the inhibitors may notreach the site of action of enzymes involved in ethylene biosynthesisand may possibly block other biosynthetic pathways which areof crucial importance to somatic embryo development. The resultsindicate that a delicate differentiation process like somaticembryogenesis is very sensitive to metabolic perturbances. Theresults are also discussed in the light of other known effectsof these four compounds on higher plants. Key words: Ethylene, Medicago sativa, somatic embryogenesis, tissue culture     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

Selection of protoclones for high yields of indole alkaloids from suspension cultures ofCatharanthus roseus and the qualitative analysis of the compounds by LC-MS

Summary To produce economically important indole alkaloids by cell culture, we have selected protoclones ofCatharanthus roseus for high yields of catharanthine and ajmalicine. Protoplasts were enzymatically isolated from suspension-cultured cells. Protoclone VPC-10 produced catharanthine at 5.9 μg/g fresh wt of cells after 10 days of culture, although the original cell line did not produce it at a level detectable by HPLC. Under the same conditions, protoclone VPC-15 produced ajmalicine at 133.6 μg/g, which was about 3 times the productivity of the original cell line. In addition, the indole alkaloids were qualitatively confirmed by LC-MS.     

Responses of Commelina communis Stomata In Vitro

Weyers, J. D. B. and Paterson, N. W. 1987. Responses of Commelinacommunis stomata in vitro.— J. exp. Bot. 38: 631–641. Analysis of the kinetics of movements of Commelina communisL. stomata in vitro revealed a sequence of opening and closingphases dependent on the incubation medium used and the physiologicalstate of the plant material. In buffer containing 50 mol m ^–3KC1 the sequence of aperture changes appeared to be influencedby equilibration of cell water potentials with that of the mediumand by solute fluxes (dependent and independent on metabolicactivity). The results indicate that the stomatal aperture afterseveral hours of incubation may not always provide a reliablequantitative estimate of the ability of the stomata to operate.As a consequence, modifications are suggested to the ways inwhich experiments using epidermal strips are carried out andreported. Key words: Epidermal strips, guard cells, hydroactive, hydropassive, kinetics, potassium chloride, mannitol, osmotic effects, solute fluxes.     

Microelectrophoretic and 9-Aminoacridine Fluorescence Study of the Surface Electrical Properties of Suspension Cultured Catharanthus roseus Cells and Isolated Protoplasts: Effects of Abscisic Acid Treatment

The effects of abscisic acid (ABA) treatments on the surfaceelectrical properties of cells and isolated protoplasts fromCatharanthus roseus cell suspension cultures were studied byelectrophoretic mobility and 9-aminoacridine (9AA) fluorescencemeasurements. The surface charge densities of the cells andprotoplasts estimated from electrokinetic data were –0.064Cm^–2and –0.048 C m^–2 respectively. These values wereclose to that estimated by 9AA fluorescence technique i.e.,–0.053 Cm^–2 for the cells and –0.041 Cm^–2for the isolated protoplasts accordingly. The net negative surfacecharge density decreased after application of 10 µM and50 µM ABA in both cells and protoplats, the more pronouncedeffect being observed at 10 µM ABA. When 100 µMABA was supplemented to the cell suspension culture the oppositeeffect was observed. The average charge density increased to–0.074 C m^–2 for the cells, and to –0.055C m^–2 for protoplasts, as revealed from the 9AA measurements.The results are discussed in terms of specific concentrationdependent ABA-induced alterations of the electrostatic propertiesof cell and protoplast membranes. (Received December 12, 1994; Accepted April 3, 1995)     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

The Relationship between Growth Rate, Differentiation and Alkaloid Accumulation in Cell Cultures

The relationship between alkaloid content, growth rate and differentiationwas investigated in seven solanaceous species in culture. Evidencewas obtained for an inverse correlation between the growth phaseon the one hand and alkaloid content and cell organization (aggregation)on the other. Linear phase and stationary phase cells of Daturainnoxia Mill, incorporated DL-(1_–14C)ornithine-HCl differentiallyinto protein and alkaloids with proportionately more label foundin alkaloids in the stationary phase cells. On transferringcallus of D. innoxia from high auxin medium (containing 10–6M 2, 4–D) to low auxin medium (containing 10–6 M,2, 4–D), the alkaloid content dropped concomitant witha decrease in chlorophyll content and an increase in growthrate and friability. The nature of the interrelationships betweengrowth, differentiation and accumulation are considered. Key words: Tropane alkaloids, Accumulation, Growth, Differentiation     

Glutamine Metabolism in Neurospora crassa under Conditions of Copper Toxicity

Rao, S. and Venkateswerlu, G. 1986. Glutamine metabolism inNeurospora crassa under conditions of copper toxicity.—J.exp. Bot. 37: 947–955. The enzyme, glutamine synthetase, of Neurospora crassa was inhibitedby copper in a non-competitive manner. Nitrate reductase activityincreased with an increase in copper concentration in the culturemedium probably as a consequence of decreased glutamine synthetaseactivity. The hexosamine content was low, whereas DNA and RNAcontents were high in cultures of N. crassa inhibited by copper.A slight accumulation of arginine and 20% less arginase activitywere observed in such cultures. Iron counteracted the toxicityof copper. Key words: Glutamine metabolism, copper toxicity, Neurospora crassa, glutamine synthetase