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.     

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.     

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.     

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     

Synthesis and Antiviral Evaluation of N-β-D-Ribosides of Ergot Alkaloids

Abstract

N-β-D-Ribosides of agroclavine (1), elymoclavine (2), lysergene (4), lysergol (3), and 9, 10-dihydrolysergol (5) were prepared by SnCl₄ catalyzed ribosylation of their TMS derivatives with 1-O-acetyl-2, 3,5-tri-O-benzoyl-β-D-ribofuranose. None of the new compounds exhibited activity against HIV or other viruses tested.     

Separation of ergot alkaloids by adsorption on silicates

A mixture of ergot alkaloids (agroclavine, elymoclavine, chanoclavine, and chanoclavine aldehyde) was separated from the Claviceps purpureafermentation broth by adsorption on inorganic adsorbents containing silica. The uptake of alkaloids depended on the concentration of adsorbent and pH. The adsorption capacity for of inorganic materials increased with increasing content of inorganic oxides such as MgO and CaO in the adsorbent. Using statistical thermodynamics, a simple mathematical model describing the multicomponent adsorption equilibrium is proposed and a numerical method suitable for fast computer simulation of multicomponent adsorption was developed.     

Effect of cultivation temperature,clomiphene, and nystatin on the oxidation and cyclization of chanoclavine in submerged cultures of the mutant strainclaviceps purpurea 59

Submerged cultures ofClaviceps purpurea 59 produce predominantly tricyclic chanoclavine and chanoclavine-I-aldehyde; formation of the tetracyclic agroclavine and elymoclavine is limited to 20%. The production cultures were characterized by the oxidation index o_i (100% chanoclavine/% chanoclavine) and cyclization index c_i (% agroclavine +% elymoclavine/% chanoclavine-I-aldehyde), expressing two functions of chanoclavine cyclase. Both indices were influenced by cultivation temperature and membrane agents. At 20°–24°C, clomiphene increased o_i and c_i; at 28°C and more it increased o_i only. At 24°C nystatin increased o_i and decreased c_i. During cultivation of vegetative inocula and production cultures at various temperatures (24°, 28°, and 33°C), the lower cultivation temperature of the inoculum increased c_i of the production culture with the higher temperature. The higher cultivation temperature of the inoculum decreased o_i and particularly c_i of the production culture with the lower temperature. In production cultures cultivated from the inoculum of an identical temperature, o_i decreased to 50% with increasing temperature above 24°C, whereas c_i decreased continuously. The role of the chanoclavine cyclase conformation as a membrane enzyme is discussed.     

Comparison of ergot alkaloid biosynthesis gene clusters in Claviceps species indicates loss of late pathway steps in evolution of C. fusiformis

The grass parasites Claviceps purpurea and Claviceps fusiformis produce ergot alkaloids (EA) in planta and in submerged culture. Whereas EA synthesis (EAS) in C. purpurea proceeds via clavine intermediates to lysergic acid and the complex ergopeptines, C. fusiformis produces only agroclavine and elymoclavine. In C. purpurea the EAS gene (EAS) cluster includes dmaW (encoding the first pathway step), cloA (elymoclavine oxidation to lysergic acid), and the lpsA/lpsB genes (ergopeptine formation). We analyzed the corresponding C. fusiformis EAS cluster to investigate the evolutionary basis for chemotypic differences between the Claviceps species. Other than three peptide synthetase genes (lpsC and the tandem paralogues lpsA1 and lpsA2), homologues of all C. purpurea EAS genes were identified in C. fusiformis, including homologues of lpsB and cloA, which in C. purpurea encode enzymes for steps after clavine synthesis. Rearrangement of the cluster was evident around lpsB, which is truncated in C. fusiformis. This and several frameshift mutations render CflpsB a pseudogene (CflpsB(Psi)). No obvious inactivating mutation was identified in CfcloA. All C. fusiformis EAS genes, including CflpsB(Psi) and CfcloA, were expressed in culture. Cross-complementation analyses demonstrated that CfcloA and CflpsB(Psi) were expressed in C. purpurea but did not encode functional enzymes. In contrast, CpcloA catalyzed lysergic acid biosynthesis in C. fusiformis, indicating that C. fusiformis terminates its EAS pathway at elymoclavine because the cloA gene product is inactive. We propose that the C. fusiformis EAS cluster evolved from a more complete cluster by loss of some lps genes and by rearrangements and mutations inactivating lpsB and cloA.     

Laboratory production of ergot alkaloids by species of balansia.

Four species of Balansia (clavicipitaceous systemic grass pathogens) isolated from pastures where cattle showed signs of ergot toxicity were grown in culture. Balansia epichlo?, one isolate of B. claviceps, B. henningsiana and two isolates of B. strangulans produced conidia in submerged culture during the first stage of a two-stage fermentation procedure. When tranferred to a glucose/sorbitol/inorganic salts medium during the second stage, these four species produced ergot alkaloids in stationary cultures. The transfer of fungi cultured in the first medium to the second medium was necessary for alkaloid biosynthesis. One isolate of B. claviceps did not produce alkaloids. Balansia epichlo? produced chanoclavine (I), agroclavine, penniclavine, elymoclavine, ergonovine and ergonovinine. Balansia claviceps produced chanoclavine (I), ergonovine and ergonovinine. This is the first report of isolating ergonovine and ergonovinine, two lysergic acid derivatives, from fungi outside the genus Claviceps. Only chanoclavine (I) was identified from extracts of B. strangulans and B. henningsiana. Chanoclavine (I) and ergonovine were identified from smut grass (Sporobolus poiretii) parasitized by B. epichlo?, indicating that this endophyte produces alkaloids both in vivo and in vitro.     

Overproduction of medicinal ergot alkaloids based on a fungal platform

Privileged ergot alkaloids (EAs) produced by the fungal genus Claviceps are used to treat a wide range of diseases. However, their use and research have been hampered by the challenging genetic engineering of Claviceps. Here we systematically refactored and rationally engineered the EA biosynthetic pathway in heterologous host Aspergillus nidulans by using a Fungal-Yeast-Shuttle-Vector protocol. The obtained strains allowed the production of diverse EAs and related intermediates, including prechanoclavine (PCC, 333.8 mg/L), chanoclavine (CC, 241.0 mg/L), agroclavine (AC, 78.7 mg/L), and festuclavine (FC, 99.2 mg/L), etc. This fungal platform also enabled the access to the methyl-oxidized EAs (MOEAs), including elymoclavine (EC), lysergic acid (LA), dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA), by overexpressing a P450 enzyme CloA. Furthermore, by optimizing the P450 electron transfer (ET) pathway and using multi-copy of cloA, the titers of EC and DHLG have been improved by 17.3- and 9.4-fold, respectively. Beyond our demonstration of A. nidulans as a robust platform for EA overproduction, our study offers a proof of concept for engineering the eukaryotic P450s-contained biosynthetic pathways in a filamentous fungal host.     

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.     

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.     

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.     

Minor alkaloids from the fungus Penicillium roquefortii Thom 1906

New spatial of clavine alkaloids, distinguished by low chromatographical mobility, have been isolated from the collection and mutant strain of Penicillium roquefortii, in addition to alkaloids roquefortine, 3,12-dihydroroquefortine, isofumigaclavines A and B, festuclavine, and chanoclavine-I, characteristic of this fungal species. In has been shown that the collection strain produces isomers of agroclavine and epoxyagroclavine, and the mutant strain produces isomers of fumigaclavines A and B, festuclavine, and chanoclavine.     

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.     

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.     

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids.

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.     

Comparison of Ergot Alkaloid Biosynthesis Gene Clusters in Claviceps Species Indicates Loss of Late Pathway Steps in Evolution of C. fusiformis

The grass parasites Claviceps purpurea and Claviceps fusiformis produce ergot alkaloids (EA) in planta and in submerged culture. Whereas EA synthesis (EAS) in C. purpurea proceeds via clavine intermediates to lysergic acid and the complex ergopeptines, C. fusiformis produces only agroclavine and elymoclavine. In C. purpurea the EAS gene (EAS) cluster includes dmaW (encoding the first pathway step), cloA (elymoclavine oxidation to lysergic acid), and the lpsA/lpsB genes (ergopeptine formation). We analyzed the corresponding C. fusiformis EAS cluster to investigate the evolutionary basis for chemotypic differences between the Claviceps species. Other than three peptide synthetase genes (lpsC and the tandem paralogues lpsA1 and lpsA2), homologues of all C. purpurea EAS genes were identified in C. fusiformis, including homologues of lpsB and cloA, which in C. purpurea encode enzymes for steps after clavine synthesis. Rearrangement of the cluster was evident around lpsB, which is truncated in C. fusiformis. This and several frameshift mutations render CflpsB a pseudogene (CflpsB^Ψ). No obvious inactivating mutation was identified in CfcloA. All C. fusiformis EAS genes, including CflpsB^Ψ and CfcloA, were expressed in culture. Cross-complementation analyses demonstrated that CfcloA and CflpsB^Ψ were expressed in C. purpurea but did not encode functional enzymes. In contrast, CpcloA catalyzed lysergic acid biosynthesis in C. fusiformis, indicating that C. fusiformis terminates its EAS pathway at elymoclavine because the cloA gene product is inactive. We propose that the C. fusiformis EAS cluster evolved from a more complete cluster by loss of some lps genes and by rearrangements and mutations inactivating lpsB and cloA.     

Glycosylation of ergot alkaloids by free and immobilized cells of Claviceps purpurea

Summary A new strain, Claviceps purpurea 88-EP-47, with high invertase activity was selected. Free and Calginate immobilized cultures of this strain were used for fructosylation of ergot alkaloids. By bioconversion from their aglycones, elymoclavine-O--d-fructofuranosyl(21)-O--d-fructofuranoside, and elymoclavine-O--d-fructoside, the respective fructosides of chanoclavine, lysergol and dihydro-lysergol monofructosides were obtained. These substances are formed by -d-fructofuranosidase present in Claviceps cells. The bioconversion activity of the enzyme system (fructose transfer) is strongly dependent on pH, substrate (sucrose) concentration and the developmental profile of invertase activity. The pH optimum for elymoclavine fructosylation is 6.5, for chanoclavine 5.7, and the optimal sucrose concentration is 75 g/l. Fifteen-day-old production cultures had the best glycosylation activities. Fructosylation of alkaloids can be stimulated in production cultures of C. purpurea or C. fusiformis forming elymoclavine or chanoclavine by a pH shift to 6.5 at the end of the production phase. Glycosylating Claviceps strains producing elymoclavine eliminate the free alkaloid into glycosides. The feedback inhibition of alkaloid synthesis by elymoclavine is then strongly reduced, helping to further improve elymoclavine yields. Elymoclavine can be liberated simply by invertase activity of baker's yeast.Offprint requests to: V. Ken     

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.