Optimization of Conditions for Storage and Cultivation of the Fungus Claviceps sp., a Producer of the Ergot Alkaloid Agroclavine

Conditions of agroclavine biosynthesis by the mutant Claviceps sp. strain c106 were studied. The content of agroclavine was maximum (1.5–2 g/l) on days 15–16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract. Agroclavine was the major component of the alkaloid fraction (90–95%). Storage of the culture at –70°C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.     

Disruption of spatial memory in mice exposed to agroclavine

The results of the study of the mnemotropic activity of the ergot alkaloid agroclavine are presented. Effects of this substance administered in doses of 1, 10, 25, 50, and 200 micrograms/kg on learning and spatial memory were studied in a Morris water maze. Agroclavine had no effect on learning but sharply impaired the retention. This memory impairment persisted for 48 h after the agroclavine administration. Agroclavine treatment did not affect the ability of mice to learn and retain a new skill. Possible mechanisms of the agroclavine effect on memory are discussed.     

Selection of ergot alkaloid producers by induced mutagenesis

Using the induced mutagenesis technique, A series of genetically modified Claviceps sp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Selection of Ergot Alkaloid Producers by Induced Mutagenesis

Using the induced mutagenesis technique, a series of genetically modified Clavicepssp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Effect of agroclavine on NK activity in vivo under normal and stress conditions in rats.

Agroclavine is a natural, clavine type of ergot alkaloid with D1 dopamine and a-adrenoceptor agonistic properties. We showed previously that in vitro agroclavine enhances natural killer (NK) cell activity, increases interleukin-2 and interferon-gamma production and prolongs the survival time of tumor-bearing mice. The aim of this study was 1) to test the effect of agroclavine on NK activity in vivo, and 2) to assess the potential toxicity of high doses of agroclavine on cardiac and liver functions using creatine kinase MB (CKMB) and alanine aminotransferase (ALT) as biochemical markers in normal and stressed animals. The effect of stress was studied because we examined promising anticancer properties of agroclavine and malignant diseases are supposed to be a potent stressful event for patients. In our experiments 3-month-old male rats of the Wistar-Kyoto strain were used. Agroclavine was injected intraperitoneally (0.5 mg/kg or 0.05 mg/kg) 30 min before stress (four hours' restraint and immersion in 23 degrees C water). The animals were killed 30 min after stress, blood was collected and the spleen was removed. Non-stressed animals treated with agroclavine were killed 5 h after the drug administration. The results confirmed our previous in vitro results and showed that also in vivo agroclavine increases NK cell activity under non-stress conditions. Agroclavine only slightly increased CKMB and had no influence on ALT in non-stressed animals. These promising results are limited by the fact that agroclavine (0.5 mg/kg) diminished NK cell activity and significantly increased ALT and CKMB under stress conditions.     

Studies onClaviceps parasitic onPanicum species in India

Panicum repens andP. antidotale were found to be infected withClaviceps sp. This is the first report of ergot onP. repens. The pyrenomycete produced abundant sclerotia on the host plants. The sclerotia contained 0.71 and 0.68 % alkaloids, respectively, which predominantly consisted of chanoclavine, festuclavine and agroclavine. The infected grasses were possibly mycotoxic. Submerged cultures ofClaviceps strain isolated fromPanicum spp produced significant amount of chanoclavine, festuclavine and agroclavine. No pharmaceutically important alkaloid was found in sclerotia or in submerged culture.     

Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

Different lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungus Epichloë festucae var. lolii × Epichloë typhina isolate Lp1 (henceforth referred to as Epichloë sp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate genes easH and cloA were expressed in a mutant strain of the mold Aspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with the Epichloë sp. Lp1 allele of easA, which encodes an enzyme that catalyzed the synthesis of agroclavine from an A. fumigatus intermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressing easA and cloA from Epichloë sp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result of Epichloë sp. Lp1 easA expression in the absence of cloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.     

Incorporation of L-[U-14C]leucine into ergosine by cell-free extracts of Clavicepspurpurea (FR.) tul

A cell-free system of $\text{Claviceps}\text{purpurea}$ has been described which incorporates [¹⁴C] leucine into the peptide-type ergot alkaloid ergosine. Cell-free extracts may be prepared either from protoplasts or lyophilized mycelium. Production was markedly stimulated by addition of agroclavine compared with d-lysergic acid and by agitation of the incubation mixture. The synthesis of ergosine is strongly dependent on the presence of ATP in the reaction mixture.     

Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

Summary Kinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both elymoclavine-inhibitionless and clavine alkaloid-decompositionless fermentations if a combination of fermentation and separation units in a closed loop was used.Nomenlacture k ₁ rate constant of agroclavine synthesis (mg Agro · mg Elymo/l·g DW·day for stage 1, mg Agro/g DW·day for stage 2) - k ₂ parameter describing inhibition of agroclavine formation rate by elymoclavine (mg Elymo/l) - k ₃ specific rate of agroclavine decay (l/g DW·day) - k ₄ maximal specific rate of elymoclavine synthesis (stage 1, 1/g DW·day, stage 2, mg Elymo/g DW·day) - k ₄ ^– maximal specific rate of elymoclavine synthesis in stage 1 (inhibition-activation mechanism) (mg Elymo/g DW·day) - k ₅ physiological constant describing the elymoclavine decay rate (l²/g DW·day·mg Elymo) - k ₅ ^– physiological constant describing the activation of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₆ physiological constant describing the repression of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₇ maximal specific growth rate (1/day) - k ₈ specific rate of biomass decay (l/g DW·day) - A agroclavine concentration (mg/l) - E elymoclavine concentration (mg/l) - r _A specific rate of agroclavine biosynthesis (mg Agro/g DW·day) - r _E specific rate of elymoclavine biosynthesis (mg Elymo/g DW·day) - r _i specific rate of alkaloid biosynthesis (mg alkaloid/g DW·day) - X dry biomass concentration (g/l) - specific growth rate (1/day) Abbreviations Agro agroclavine - Elymo elymoclavine - Chano chanoclavine - DW dry weight of biomass     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

Analysis of secondary metabolites of microscopic fungi of the genus Penicillium by chromatographic techniques

Combinations of various regimens of thin-layer chromatography and high-performance liquid chromatography (HPLC) was efficient in analyzing 39 nitrogen-containing secondary metabolites (alkaloids) produced by 12 strains of microscopic fungi of the genus Penicillium. Chromatographic mobility of alkaloids on Silufol plates was determined in the following systems (following staining with the Ehrlich reagent): (a) chloroform, methanol, and 25% NaOH (90:10:1, 90:10:0.1, or 80:20:0.2); (b) chloroform and acetone (9:1); and (c) ethyl acetate, methanol, and 25% NH4OH (85:15:10). Conditions for separation of clavine alkaloids by HPLC on Spherisorb ODS2 and Supelcosil LC-18 columns (gradient elution) were optimized. Retention values of 22 alkaloids were compared to those of agroclavine and roquefortine.     

Analysis of Secondary Metabolites of Microscopic Fungi of the Genus Penicillium by Chromatographic Techniques

Combinations of various systems of thin-layer chromatography and high-performance liquid chromatography (HPLC) were efficient in analyzing 39 nitrogen-containing secondary metabolites (alkaloids) produced by 12 strains of microscopic fungi of the genus Penicillium. Chromatographic mobility of alkaloids on Silufol plates was determined in the following systems: (a) chloroform, methanol, and 25% NH₄OH (90 : 10 : 1, 90 : 10 : 0.1, or 80 : 20 : 0.2); (b) chloroform and acetone (9 : 1); and (c) ethyl acetate, methanol, and 25% NH₄OH (85 : 15 : 10); staining was performed using Ehrlich's reagent. Conditions for separation of clavine alkaloids by HPLC on Spherisorb ODS-2 and Supelcosil LC-18 columns (gradient elution) were optimized. Retention values of 22 alkaloids were compared to those of agroclavine and roquefortine.     

A study of the modified type II spectral change produced by the interaction of agroclavine with cytochrome P-450

Previous work in this laboratory has demonstrated that agroclavine is metabolized by liver microsomes and that cytochrome P-₄₅₀ is involved as the terminal oxidase in the reaction. Addition of agroclavine to suspensions of liver microsomes in the same concentration as used in the metabolic studies produced a modified Type II spectral change with peak near 420 nm and trough near 390 nm. Using modifiers such as hexobarbital, this spectrum has now been shown to contain a Type 1 component. Furthermore, when sufficiently low concentrations of agroclavine were added to rat liver microsomes, a Type I spectral change appeared, which gradually turned into the modified Type II variety upon increase in agroclavine concentration.     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine     

Optimization of culture conditions for production of the anti-tubercular alkaloid hirsutellone A by Trichoderma gelatinosum BCC 7579

AIMS: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. METHODS AND RESULTS: The fungus was initially cultured in shake flasks at 25 degrees C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l(-1), obtained from basal condition, to 610.55 mg l(-1) and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min(-1) at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l(-1) of hirsutellone A within 6 days. CONCLUSIONS: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with an initial pH of 7. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future.     

Effects of clavines, ergot alkaloids, on the membrane potential of an identifiable giant neuron of the African giant snail Achatina fulica Ferussac

The effects of seven clavines, alkaloids of ergot, on the electrical activity of an identifiable giant neurone (TAN, tonically autoactive neurone) of the African giant snail were examined. All the substances examined, lysergine, agroclavine, elymoclavine, festuclavine, chanoclavine, rugulovasine A and rugulovasine B, at 2 X 10(-4) kg/l have no constant effect on TAN, indicating that they have no direct effect on this neurone. However, the substances examined, except for chanoclavine, in the same concentration occasionally caused the transient depression with an augmentation of trans-synaptic influences. This depression may be due to the trans-synaptic influences. The four substances examined, lysergine, agroclavine, elymoclavine and festuclavine, in the same concentration produced TAN abnormal spike discharges, doublet or triplet spikes.     

Increase in the indole alkaloid production and its excretion into the culture medium by calcium antagonists inCatharanthus roseus hairy roots

Treatment of Catharanthus roseus hairy roots with antagonists, like verapamil and CdCl₂, that block the Ca²⁺ flux across the plasma membrane enhanced the total alkaloid content by 25% and their secretion 10 times. The specific Ca²⁺ chelator, EGTA, stimulated 90% of the total alkaloid secretion. Treatment with inhibitors of intracellular Ca²⁺ movement, like TMB-8 and trapsigargin, enhanced the total alkaloid content by 74% and their secretion into the culture media by 4- to 6-fold. The results suggest that an inhibition of external and internal Ca²⁺ fluxes induces an increase in the indole alkaloid accumulation and secretion in C. roseus hairy roots.     

Biotransformation of ergot alkaloids by plant cell cultures with high peroxidase activity

Ergot alkaloids agroclavine and elymoclavine have been modified using plant cell cultures exhibiting high peroxidase activity. Setoclavine and isosetoclavine have been isolated from media after transformation of agroclavine on a semipreparative scale. 10-Hydroxyelymoclavine resulted from similar treatment of elymoclavine.     

Physiological studies on ergot: further studies on the induction of alkaloid synthesis by tryptophan and its inhibition by phosphate

The failure of l-leucine to stimulate ergot alkaloid production in a synthetic medium indicates that the previously observed stimulation by tryptophan and tryptophan analogues does not merely represent a nutritional effect. Tryptophan, but not mevalonate or 5-methyltryptophan, is able to overcome the inhibition of alkaloid synthesis by high levels of inorganic phosphate. Therefore, high phosphate levels seem to limit the synthesis of tryptophan; they may, in addition, prevent induction of alkaloid synthesis by preventing accumulation of tryptophan. Experiments which indicate a 2- to 3-fold temporary increase of intracellular free tryptophan and a 20- to 25-fold increase of tryptophan synthetase activity during the transition period between growth and alkaloid production phase are in agreement with the previously postulated induction of alkaloid synthesis by tryptophan. The latter experiments also indicate 4- to 6-fold repression of this enzyme by tryptophan.     

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

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