Alkaloid patterns and biosynthetic capacity of root cultures from some pyrrolizidine alkaloid producing Senecio species

Root cultures of Senecio vulgaris, S. vernalis, S. erucifolius and S. squalidus were established. The patterns of pyrrolizidine alkaloids found in these root cultures were analyzed by high-resolution GC and GC-MS and compared with the alkaloids present in the respective plants. In vitro cultured roots produce alkaloid patterns and accumulate quantities which are comparable to those found in soil grown plants. With the exception of the otonecine derivative senkirkine all pyrrolizidines accumulate as N-oxides. Only senkirkine is partially released into the medium. The cultures incorporate biosynthetic precursors, e.g. ¹⁴C-labelled putrescine or spermidine with high efficiency into the alkaloids. Senecionine N-oxide was found to be the main product of biosynthesis. Evidence is presented that senecionine N-oxide is directly transformed into senkirkine, the main alkaloid of S. vernalis root cultures.Abbreviations GC Gas chromatography - MS Mass spectroscopy - PND Phosphorous-Nitrogen-Detector - FID Flame Ionization Detector - fr.wt Fresh weight     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Tropane alkaloid production inDatura stramonium root cultures

Summary Tropane alkaloid production was studied in different root cultures ofDatura stramonium. Cultured roots were obtained with 10⁻⁶ M of indolbutyric acid. Their doubling times were from 6 to 19 days. Hyoscyamine content varied from 0.17 to 0.62% dry weight, and scopolamine content from 0.08 to 0.33% dry weight, depending on the lines. A comparison of the bioproductivity of these compounds in the pot-grown plant roots showed that it was two to three orders lower than cultured roots, and it increased one order of magnitude considering the productivity on the whole plant. Bioproductivity, growth capacity and alkaloid production stability during subsequent transfers (more than 2 yr) are reported. Only one root line (N5) showed excretion of the alkaloids to the culture medium. Characterization of three selected lines (N1, N5, and N9) showed that the highest alkaloid production is reached at the stationary phase of growth, with the exception of line N9.     

Effect of fungal homogenate,enzyme inhibitors and osmotic stress on alkaloid content of Catharanthus roseus cell suspension cultures

The addition of Aspergillus niger homogenate to Catharanthus roseus cell suspension cultures produced an increment of more than 60% in the alkaloid content of two different cell lines. The use of an inhibitor of phenylalanine ammonia lyase, i. e. cinnamic acid, along with the homogenate, resulted in an appearance of 90% of the alkaloids in the medium. Furthermore, even in the absence of fungal homogenate, there was a marked increase in the alkaloid content. The exposure of the cells to an osmotic stress produced a marked increment (320%) in the total alkaloid content. When both stress treatments were applied sequentially, an additive effect on alkaloid accumulation was observed. It was 300% higher than in cells cultured without treatment, the majority of the alkaloids found in the medium.Abbreviations BAP benzylaminopurine - CM chorismate mutase - DW dry weight - FW fresh weight - IAA indole-3-acetic acid - MS Murashige-Skoog - PAL phenylalanine ammonia lyase - PC Phillips-Collins     

Production of steroidal alkaloids by hairy roots of Solanum aviculare and the effect of gibberellic acid

Cultures of Solanum aviculare hairy roots were established after transformation with Agrobacterium rhizogenes A4. High levels of steroidal alkaloids measured as solasodine equivalents were produced in shake-flasks and bioreactor, even though relatively low concentrations are found in roots in vivo. In shake flasks the maximum alkaloid yield was 32 mg g^-1 dry weight; in a 3-1 air-driven bioreactor the yield was 29 mg g^-1. These yields represent a 5-fold increase over previous reports for in vitro production, and are comparable with levels found in the aerial parts of intact S. aviculare plants. Production of steroidal alkaloids was growth-associated. High sugar levels at stationary phase and insensitivity to increased levels of medium components suggest that root cultures were limited by oxygen mass-transfer. In Petri-dish culture with and without exogenous gibberellic acid, root length and number of root tips increased exponentially; growth proceeded with a constant length per root tip of about 35 mm. Addition of gibberellic acid enhanced growth but reduced the specific steroidal-alkaloid level. Taking into account both growth and alkaloid yield, accumulation of steroidal alkaloids was improved by about 40% at gibberellic-acid concentrations of 10 and 100 g l^-1.     

Enhanced production of tropane alkaloids in <Emphasis Type="Italic">Scopolia parviflora</Emphasis> by introducing the <Emphasis Type="Italic">PMT</Emphasis> (putrescine <Emphasis Type="Italic">N</Emphasis>-methyltransferase) gene

Summary In wild-type Scopolia parvilfora (Solanaceae) tissues, only the roots express the enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53), which is the first specific precursor of the tropane alkaloids. Moreover, the tropanane alkaloid levels were the highest in the root (0.9 mg g⁻¹ on a dry weight basis), followed by the stem and then the leaves. We metabolically engineered S. parviflora by introducing the tobacco pmt gene into its genome by a binary vector system that employs disarmed Agrobacterium rhizogenes. The kanamycin-resistant hairy root lines were shown to bear the pmt gene and to overexpress its mRNA and protein product by at least two-fold, as determined by polymerase chain reaction (PCR) and Northern and Western blottings, respectively. The transgenic lines also showed higher PMT activity and were morphologically aberrant in terms of slower growth and the production of lateral roots. The overexpression of pmt markedly elevated the scopolamine and hyoscyamine levels in the transgenic lines that showed the highest pmt mRNA and PMT protein levels. Thus, overexpression of the upstream regulator of the tropane alkaloid pathway enhanced the biosynthesis of the final product. These observations may be useful in establishing root culture systems that generate large yields of tropane alkaloids. These authors contributed equally to this paper (co-first authors).     

Establishment and characterization of photosynthetic hairy root cultures of Datura stramonium

Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O₂ evolution, CO₂ fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport.     

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.     

Sites of synthesis,translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L.

¹⁴C-Labelled alkaloid precursors (arginine, putrescine, spermidine) fed to Senecio vulgaris plants via the root system were rapidly taken up and efficiently incorporated into the pyrrolizidine alkaloid senecionine N-oxide (sen-Nox) with total incorporations of 3–6%. Considerable amounts of labelled sen-Nox were translocated into the shoot and were directed mainly into the inflorescences, the major sites of pyrrolizidine-alkaloid accumulation. Detached shoots of S. vulgaris were unable to synthesize pyrrolizidine alkaloids, indicating that the roots are the site of their biosynthesis. Further evidence was obtained from studies with in-vitro systems established from S. vulgaris: root cultures were found to synthesize pyrrolizidine alkaloids but not cell-suspension cultures, tumor cultures or shoot-like teratomas obtained by transformation with Agrobacterium tumefaciens. Studies on transport of [¹⁴C]sen-Nox, which was fed either to detached shoots or to the root system of intact plants, indicate that the alkaloid N-oxide does not simply follow the transpiration stream but is specifically channelled to the target tissues such as epidermal stem tissue and flower heads. Exogenously applied [¹⁴C]senecionine is rapidly N-oxidized. If the phloem path along the stem is blocked by a steam girdle translocation of labelled sen-Nox is blocked as well. Root-derived sen-Nox accumulated below the girdle and only trace amounts were found in the tissues above. It is most likely that the root-to-shoot transport of sen-Nox occurs mainly if not exclusively via the phloem. In accordance with previous studies the polar, salt-like N-oxides, which are often considered to be artifacts, were found to be the real products of pyrrolizidine-alkaloid biosynthesis as well as the physiological forms for long-distance transport, tissue-specific distribution and cellular accumulation.Abbreviations FW fresh weight - sen senecionine - sen-Nox senecionine N-oxide     

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

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

Overexpression of a Catharanthus tryptophan decarboxylase (tdc) gene leads to enhanced terpenoid indole alkaloid (TIA) production in transgenic hairy root lines of Rauwolfia serpentina

To enhance the production of terpenoid indole alkaloids in Rauwolfia serpentina, Catharanthus tryptophan decarboxylase (Crtdc) gene was over-expressed in transgenic hairy root cultures using Agrobacterium rhizogenes-mediated transformation. Among six transgenic hairy root lines, line RT4 accumulated the highest alkaloid content, with 0.1202 % dry weight (DW) reserpine and 0.0064 % DW ajmalicine, after 10 weeks of culture. Whereas, wild-type roots accumulated 0.0596 ± 0.003 % DW reserpine and 0.0011 ± 0.001 % DW ajmalicine. Transgenic hairy root line RT7 produced the lowest alkaloid content (reserpine: 0.0896 ± 0.002 % DW; ajmalicine: 0.002 ± 0.0 % DW). On the basis of alkaloid content the six hairy root lines were grouped as RT4/RT2 > RT3/RT5 > RT7/RT8. Analysis of gene expression profile indicated that Crtdc was expressed at a higher level in transgenic lines, which could be correlated with enhanced metabolite accumulation in roots. This study confirms that over-expression of Crtdc is a superlative method to improve the biosynthetic potential of Rauwolfia hairy root cultures. Enhanced reserpine and ajmalicine production can serve as an alternative choice to provide resources for relative pharmaceutical industries.     

Calystegines as a new group of tropane alkaloids in Solanaceae

Calystegines are a new group of polyhydroxy alkaloids with a nortropane skeleton. They were detected in Atropa belladonna root cultures by chromatographic methods (TLC, GC) and identified by NMR and mass spectroscopy. Their occurrence was examined in several species of the Solanaceae. The biosynthesis of these compounds is suggested to proceed via the tropane alkaloid pathway, the first metabolite being pseudotropine. A pseudotropine-forming tropinone reductase was isolated and characterized from Atropa belladonna root cultures. Further evidence is given for the significance of tropinone and pseudotropine in calystegine formation by feeding experiments that increased calystegine formation. ¹⁵N-tropinone was shown to be incorporated into calystegines.Abbreviations GC gas chromatography - TBON 8-thiabicyclo[3.2.1]octan-3-one - TLC thin-layer chromatography     

Induction of tropane alkaloid formation in transformed root cultures of Brugmansia suaveolens (Solanaceae)

Hairy root cultures of Brugmansia suaveolens were set up by infection of root tips with Agrobacterium rhizogenes. The successful transformation was confirmed by analysing rolC and virC genes using polymerase chain reaction (PCR). Hairy root cultures were employed to study the formation of tropane alkaloids, such as hyoscyamine. The transformed cultures were incubated with potential elicitors, such as methyljasmonate, quercetin and salicylic acid in order to stimulate the biosynthesis of tropane alkaloids. Profile and amounts of tropane alkaloids were analysed using capillary GLC-MS. At least 18 different tropane alkaloids could be identified. Treatment of the cultures with 200 microM methyljasmonate increased the alkaloid accumulation 25-fold up to a level of 1 mg/g fresh weight as compared to untreated controls. Quercetin enhanced the alkaloid production 10 fold (0.4 mg/g fresh weight) within 24 h. In contrast 100 microM salicylic acid decreased alkaloids to a level of 1 microg/g fresh weight.     

Camptothecin and 10-hydroxycamptothecin from<Emphasis Type="Italic"> Camptotheca acuminata</Emphasis> hairy roots

Camptothecin (CPT) is an anticancer and antiviral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae) and some other species belonging to the families Apocynaceae, Olacaceae, and Rubiaceae. Bark and seeds are currently used as sources for the drug. Several attempts have been made to produce CPT from cell suspensions; however, the low yields obtained limit this approach. Cultures of differentiated cell types may be an alternative source of alkaloid production. Hairy root cultures of C. acuminata were established from tissue transformed with Agrobacterium rhizogenes strains ATCC 15834 and R-1000. Integration of the genes responsible for the hairy-root phenotype (rol genes) into the plant genome was verified by DNA gel blot analysis. The hairy roots produce and secrete CPT as well as the more potent and less toxic natural derivative, 10-hydroxycamptothecin (HCPT), into the medium. Remarkably, the cultures were able to synthesize the alkaloids at levels equal to, and sometimes greater than, the roots in planta, i.e., 1.0 and 0.15 mg/g dry weight for CPT and the HCPT, respectively.Abbreviations CPT Camptothecin - DR Dry weight - FW Fresh weight - HCPT 10-Hydroxycamptothecin - HPLC High-performance liquid chromatography - kb Kilobases - NAA 1-Napththalenacetic acid - TLC Thin-layer chromatographyCommunicated by K.K. Kamo     

CO2 enrichment and supporting material impact the primary metabolism and 20-hydroxyecdysone levels in Brazilian ginseng grown under photoautotrophy

Plant cell and organ cultures via the implementation of effective elicitation strategies can offer attractive biotechnological platforms for the enhanced production of phytochemicals of pharmaceutical interest. For the first time, the elicitation of exogenous signal molecules was conducted to enhance the production of pharmacologically active alkaloids and flavonoids in Isatis tinctoria L. hairy root cultures (ITHRCs). ITHRCs III and V correspondingly possessing high alkaloid and flavonoid productivity were adopted for elicitation treatments. The maximum accumulation of alkaloids in ITHRCs III elicited by 142.61 µM salicylic acid for 28.18 h and flavonoids in ITHRCs V elicited by 179.54 µM methyl jasmonate for 41.87 h increased 5.89- and 11.21-folds as compared with controls, respectively. Moreover, expressions of 11 genes involved in alkaloid and flavonoid biosynthetic pathways were significantly up-regulated following elicitation, among which YUCCA, CHI and F3′H genes might play a crucial role in the target phytochemical augmentation. Overall, two effective elicitation protocols were provided here to improve the yields of bioactive alkaloids and flavonoids in ITHRCs, which was useful for the scale-up production of these valuable compounds to meet the demands for natural bioactive ingredients by pharmaceutical industries.

     

Breakdown of indole alkaloids in suspension cultures of Tabernaemontana divaricata and Catharanthus roseus

The relative importance of breakdown on the accumulation of indole alkaloids has been determined in suspension cultures of Tabernaemontana divaricata and Catharanthus roseus by the feeding of stable isotope labelled alkaloids. In all cultures a considerable amount of the alkaloid biosynthesized was broken down. The breakdown was found to be dependent on the culture period and the half-life was in the order of several days. The breakdown could not explain the difference between producing and non-producing cultures. Further it was determined that in both cultures the breakdown was due to both biotic and abiotic factors.     

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     

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.     

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.     

The effect of temperature on hairy root cultures of Catharanthus roseus: Growth,indole alkaloid accumulation and membrane lipid composition

Cultivation of Catharanthus roseus hairy root cultures at different temperatures was found to have an effect on growth rate and indole alkaloid content as well as lipid composition. When lowering the temperature, the roots responded by increasing the degree of unsaturation of cellular lipids, which was mainly due to an increased proportion of linolenic acid in the main lipid classes. The modifications in lipid composition were obviously necessary for the roots to retain the proper cell membrane fluidity at each temperature. Despite of changes in membrane lipids, no effect on the distribution of indole alkaloids between the roots and the medium could be detected. Instead, the level of alkaloid accumulation showed a clear increase with lowering temperature.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PG phosphatidylglycerol - CL cardiolipin - DGD digalactosyldiglyceride - MGD monogalactosyldiglyceride - NL neutral lipids - DU degree of fatty acid unsaturation