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Irritable bowel syndrome (IBS) is a chronic disease that causes abdominal pain and an imbalance of defecation patterns due to gastrointestinal dysfunction. The cause of IBS remains unclear, but intestinal–brain axis problems and neurotransmitters have been suggested as factors. In this study, chanoclavine, which has a ring structure similar to 5-hydroxytryptamine (5-HT), showed an interaction with the 5-HT3A receptor to regulate IBS. Although its derivatives are known to be involved in neurotransmitter receptors, the molecular physiological mechanism of the interaction between chanoclavine and the 5-HT3A receptor is unknown. Electrophysiological experiments were conducted using a two-electrode voltage-clamp analysis to observe the inhibitory effects of chanoclavine on Xenopus oocytes in which the h5-HT3A receptor was expressed. The co-application of chanoclavine and 5-HT resulted in concentration-dependent, reversible, voltage-independent, and competitive inhibition. The 5-HT3A response induced by 5-HT was blocked by chanoclavine with half-maximal inhibitory response concentration (IC50) values of 107.2 µM. Docking studies suggested that chanoclavine was positioned close F130 and N138 in the 5-HT3A receptor-binding site. The double mutation of F130A and N138A significantly attenuated the interaction of chanoclavine compared to a single mutation or the wild type. These data suggest that F130 and N138 are important sites for ligand binding and activity. Chanoclavine and ergonovine have different effects. Asparagine, the 130th amino acid sequence of the 5-HT3A receptor, and phenylalanine, the 138th, are important in the role of binding chanoclavine, but ergonovine has no interaction with any amino acid sequence of the 5-HT3A receptor. The results of the electrophysiological studies and of in silico simulation showed that chanoclavine has the potential to inhibit the hypergastric stimulation of the gut by inhibiting the stimulation of signal transduction through 5-HT3A receptor stimulation. These findings suggest chanoclavine as a potential antiemetic agent for excessive gut stimulation and offer insight into the mechanisms of 5-HT3A receptor inhibition.

An efficient and direct route to ergot alkaloid (-)-chanoclavine I (3) is described using the inexpensive compound (2R)-(+)-phenyloxirane (15) as a chiral pool in 13 steps with 17% overall yield. Key features of the synthesis include a palladium-catalyzed intramolecular aminoalkynylation of terminal olefin and a rhodium-catalyzed intramolecular [3 + 2] annulation. An oxygen-substituted ergoline derivative (-)-25 was also achieved by using the same strategy.

Epichlo&euml, endophytes have been used successfully in pastoral grasses providing protection against insect pests through the expression of secondary metabolites. This approach could be extended to other plant species, such as cereals, reducing reliance on pesticides. To be successful, the selected endophyte must express secondary metabolites that are active against cereal insect pests without any secondary metabolite, which is harmful to animals. Chanoclavine is of interest as it is commonly expressed by endophytes and has potential insecticidal activity. Investigation of possible mammalian toxicity is therefore required. An acute oral toxicity study showed the median lethal dose of chanoclavine to be &gt, 2000 mg/kg. This allows it to be classified as category 5 using the globally harmonized system of classification and labelling of chemicals, and category 6.1E using the New Zealand Hazardous Substances and New Organisms (HSNO) hazard classes, the lowest hazard class under both systems of classification. A three-week feeding study was also performed, which showed chanoclavine, at a dose rate of 123.9 mg/kg/day, initially reduced food consumption but was resolved by day seven. No toxicologically significant effects on gross pathology, histology, hematology, or blood chemistry were observed. These experiments showed chanoclavine to be of low toxicity and raised no food safety concerns.

Epichlo&euml, endophytes are filamentous fungi (family Clavicipitaceae) that live in symbiotic associations with grasses in the sub family Po&ouml, ideae. In New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and is an essential component of pastoral agriculture, where ryegrass is a major forage species. The fungus produces in planta a range of bioactive secondary metabolites, including ergovaline, which has demonstrated bioactivity against the important pasture pest black beetle, but can also cause mammalian toxicosis. We genetically modified E. festucae var. lolii strain AR5 to eliminate key enzymatic steps in the ergovaline pathway to determine if intermediate ergot alkaloid compounds can still provide insecticidal benefits in the absence of the toxic end product ergovaline. Four genes (dmaW, easG, cloA, and lpsB) spanning the pathway were deleted and each deletion mutant was inoculated into five different plant genotypes of perennial ryegrass, which were later harvested for a full chemical analysis of the ergot alkaloid compounds produced. These associations were also used in a black beetle feeding deterrence study. Deterrence was seen with just chanoclavine present, but was cumulative as more intermediate compounds in the pathway were made available. Ergovaline was not detected in any of the deletion associations, indicating that bioactivity towards black beetle can be obtained in the absence of this mammalian toxin.

Ergot alkaloids are pharmaceutically and agriculturally important secondary metabolites produced by several species of fungi. Ergot alkaloid pathways vary among different fungal lineages, but the pathway intermediate chanoclavine-I is evolutionarily conserved among ergot alkaloid producers. At least four genes, dmaW, easF, easE, and easC, are necessary for pathway steps prior to chanoclavine-I; however, the sufficiency of these genes for chanoclavine-I synthesis has not been established. A fragment of genomic DNA containing dmaW, easF, easE, and easC was amplified from the human-pathogenic, ergot alkaloid-producing fungus Aspergillus fumigatus and transformed into Aspergillus nidulans, a model fungus that does not contain any of the ergot alkaloid synthesis genes. HPLC and LC-MS analyses demonstrated that transformed A. nidulans strains produced chanoclavine-I and an earlier pathway intermediate. Aspergillus nidulans transformants containing dmaW, easF, and either easE or easC did not produce chanoclavine-I but did produce an early pathway intermediate and, in the case of the easC transformant, an additional ergot alkaloid-like compound. We conclude that dmaW, easF, easE, and easC are sufficient for the synthesis of chanoclavine-I in A. nidulans and expressing ergot alkaloid pathway genes in A. nidulans provides a novel approach to understanding the early steps in ergot alkaloid synthesis.

The putative gene fgaMT was identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus. The coding region of fgaMT was amplified by PCR from a cDNA library, cloned into pQE60, and overexpressed in Escherichia coli. FgaMT comprises 339 amino acids with a molecular mass of about 38.1 kDa. The soluble dimeric His(6)-FgaMT was purified to near homogeneity and characterized biochemically. FgaMT was found to catalyze the N-methylation of 4-dimethylallyltryptophan in the presence of S-adenosylmethionine, resulting in the formation of 4-dimethylallyl-l-abrine, which was identified by NMR and mass spectrometry analysis. Therefore, FgaMT represents the second pathway-specific enzyme in the biosynthesis of ergot alkaloids. The enzyme did not require metal ions for its enzymatic reaction and showed a relatively high specificity toward the prenyl moiety at position C-4 of the indole ring. 4-Dimethylallyltryptophan derivatives with modification at the indole ring were also accepted by FgaMT as substrates. K(m) values for 4-dimethylallyltryptophan and S-adenosylmethionine were determined at 0.12 and 2.4 mm, respectively. The turnover number was 2.0 s(-1).

The ergot alkaloid biosynthesis system has become an excellent model to study evolutionary diversification of specialized (secondary) metabolites. This is a very diverse class of alkaloids with various neurotropic activities, produced by fungi in several orders of the phylum Ascomycota, including plant pathogens and protective plant symbionts in the family Clavicipitaceae. Results of comparative genomics and phylogenomic analyses reveal multiple examples of three evolutionary processes that have generated ergot-alkaloid diversity: gene gains, gene losses, and gene sequence changes that have led to altered substrates or product specificities of the enzymes that they encode (neofunctionalization). The chromosome ends appear to be particularly effective engines for gene gains, losses and rearrangements, but not necessarily for neofunctionalization. Changes in gene expression could lead to accumulation of various pathway intermediates and affect levels of different ergot alkaloids. Genetic alterations associated with interspecific hybrids of Epichloë species suggest that such variation is also selectively favored. The huge structural diversity of ergot alkaloids probably represents adaptations to a wide variety of ecological situations by affecting the biological spectra and mechanisms of defense against herbivores, as evidenced by the diverse pharmacological effects of ergot alkaloids used in medicine.

The ergot alkaloids, a class of fungal‐derived natural products with important biological activities, are derived from a common intermediate, chanoclavine‐I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast‐based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine‐I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L−1, thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.

A putative dimethylallyltryptophan synthase gene,fgaPT2, was identified in the genome sequence ofAspergillus fumigatus.fgaPT2was cloned and overexpressed inSaccharomyces cerevisiae. The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convertl-tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD.Kmvalues forl-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 μM, respectively. Metal ions, such as Mg2+and Ca2+, enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.

Seeds from the Hawaiian Baby Woodrose Argyreia nervosa of different origin and labelling and with allegedly high levels of ergot alkaloids were analysed using high performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS/MS) technique. Lysergic acid amide (LSA), ergometrine, lysergol/elymoclavine/setoclavine, chanoclavine, lysergic acid and their respective stereoisomers were identified as well as penniclavine and lysergic acid α-hydroxyethylamide. In addition, methylergometrine, methysergide, and lysergylalanine were detected, some high molecular weight ergot alkaloid derivatives and hydroxyalanine derived ergopeptide fragments were detected indicating the presence of ergopeptides in the seeds. The results of the study demonstrate that the content of ergot alkaloids in Argyreia nervosa seeds depends on the quality of the material. For a consumer the quality of the seeds is unforeseeable. For the toxicological expert it is essential to investigate not only the identity of such a confiscated seed material, but also the various ergot alkaloid constituents to assess the hazardous nature and the toxic potential of the material.

Epichloë fungal endophytes of cool season grasses are well-known to produce a range of alkaloids of benefit to the host. Some of these compounds are advantageous to agriculture due to qualities that promote pasture persistence (e.g., the loline class of alkaloids confer insect protection) while others are detrimental to the well-being of grazing livestock. The ergot alkaloids (e.g., ergovaline), produced in ryegrass and tall fescue associations, causes poor animal health in farming regions in many countries around the world and further study is required to improve our knowledge on this class of compounds. Here we present the application of a quantitative LC-MS/MS (liquid chromatography coupled to mass spectrometry) method measuring eight ergot alkaloids (chanoclavine, agroclavine, elymoclavine, lysergol, lysergic acid, ergine, lysergyl-alanine, ergovaline) produced by endophyte infected grasses, to monitor levels in individual tillers from multiple plants of a single cultivar of perennial ryegrass (Lolium perenne cv. “Grasslands Samson”) infected with a common toxic endophyte strain (Epichloë festucae var. lolii). Monitoring the expression in individual tillers allows an estimation of the variability within a plant (between tillers) as well as between plants. The study showed that there is significant variation in the concentration of the ergot alkaloids between tillers of a single plant, at or exceeding the level of variation observed between individual plants of a population. This result emphasizes the fundamental importance of robust experimental design and sampling procedures when alkaloid expression assessment is required and these need to be rigorously tailored to the hypothesis being tested.

Epichloid endophytes are well known symbionts of many cool-season grasses that may alleviate environmental stresses for their hosts. For example, endophytes produce alkaloid compounds that may be toxic to invertebrate or vertebrate herbivores. Achnatherum robustum, commonly called sleepygrass, was aptly named due to the presence of an endophyte that causes toxic effects to livestock and wildlife. Variation in alkaloid production observed in two A. robustum populations located near Weed and Cloudcroft in the Lincoln National Forest, New Mexico, suggests two different endophyte species are present in these populations. Genetic analyses of endophyte-infected samples revealed major differences in the endophyte alkaloid genetic profiles from the two populations, which were supported with chemical analyses. The endophyte present in the Weed population was shown to produce chanoclavine I, paspaline, and terpendoles, so thus resembles the previously described Epichloe funkii. The endophyte present in the Cloudcroft population produces chanoclavineI, ergonovine, lysergic acid amide, and paspaline, and is an undescribed endophyte species. We observed very low survival rates for aphids feeding on plants infected with the Cloudcroft endophyte, while aphid survival was better on endophyte infected plants in the Weed population. This observation led to the hypothesis that the alkaloid ergonovine is responsible for aphid mortality. Direct testing of aphid survival on oat leaves supplemented with ergonovine provided supporting evidence for this hypothesis. The results of this study suggest that alkaloids produced by the Cloudcroft endophyte, specifically ergonovine, have insecticidal properties.

Background Ergot alkaloids are a group of highly bioactive molecules produced by a number of filamentous fungi. These compounds have been intensely studied for decades, mainly due to their deleterious effects in contaminated food and feeds, but also for their beneficial pharmaceutical and agricultural applications. Biosynthesis of ergot alkaloids goes via the common intermediate chanoclavine-I, and studies of the key enzymes, EasE and EasC, involved in chanoclavine-I formation, have relied on gene complementation in fungi, whereas further characterization has been hampered by difficulties of poor EasE protein expression. In order to facilitate the study of ergot alkaloids, and eventually move towards commercial production, the early steps of the biosynthetic pathway were reconstituted in the unicellular yeast Saccharomyces cerevisiae. Results The genomic sequence from an ergot alkaloid producer, Aspergillus japonicus, was used to predict the protein encoding sequences of the early ergot alkaloid pathway genes. These were cloned and expressed in yeast, resulting in de novo production of the common intermediate chanoclavine-I. This allowed further characterization of EasE and EasC, and we were able to demonstrate how the N-terminal ER targeting signal of EasE is crucial for activity in yeast. A putative, peroxisomal targeting signal found in EasC was shown to be nonessential. Overexpression of host genes pdi1 or ero1, associated with disulphide bond formation and the ER protein folding machinery, was shown to increase chanoclavine-I production in yeast. This was also the case when overexpressing host fad1, known to be involved in co-factor generation. Conclusions A thorough understanding of the enzymatic steps involved in ergot alkaloid formation is essential for commercial production and exploitation of this potent compound class. We show here that EasE and EasC are both necessary and sufficient for the production of chanoclavine-I in yeast, and we provide important new information about the involvement of ER and protein folding for proper functional expression of EasE. Moreover, by reconstructing the chanoclavine-I biosynthetic pathway in yeast we demonstrate the advantage and potential of this host, not only as a convenient model system, but also as an alternative cell factory for ergot alkaloid production. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0095-2) contains supplementary material, which is available to authorized users.

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.

The ergot alkaloids elymoclavine, ergometrine and chanoclavine were α-mannosylated with α-mannosidase as catalyst. The kinetic reaction with p-nitrophenyl α-mannoside as glycosyl donor gave ca 28 % yield of chanoclavine α-mannoside, whereas the equilibrium reaction with mannose as the glycosyl donor gave ca 11 % yield. However, in the case of elymoclavine and ergometrine, higher yields of α-mannosides were obtained with the equilibrium approach (18 and 13 %).

The ergot alkaloids are a diverse class of fungal-derived indole alkaloid natural products with potent pharmacological activities. The biosynthetic intermediate chanoclavine-I aldehyde 1 represents a branch point in ergot biosynthesis. Ergot alkaloids festuclavine 2 and agroclavine 3 derive from alternate enzymatic pathways originating from the common biosynthetic precursor chanoclavine-I aldehyde 1. Here we show that while the Old Yellow Enzyme homologue EasA from the ergot biosynthetic gene cluster of Aspergillus fumigatus acts on chanoclavine-I aldehyde 1 to yield festuclavine 2, EasA from Neotyphodium lolii, in contrast, produces agroclavine 3. Mutational analysis suggests a mechanistic rationale for the switch in activity that controls this critical branch point of ergot alkaloid biosynthesis.

Ergot fungi in the genus Claviceps and several related fungal groups in the family Clavicipitaceae produce toxic ergot alkaloids. These fungi produce a variety of ergot alkaloids, including clavines as well as lysergic acid derivatives. Ergot alkaloids are also produced by the distantly related, opportunistic human pathogen Aspergillus fumigatus . However, this fungus produces festuclavine and fumigaclavines A, B, and C, which collectively differ from clavines of clavicipitaceous fungi in saturation of the last assembled of four rings in the ergoline ring structure. The two lineages are hypothesized to share early steps of the ergot alkaloid pathway before diverging at some point after the synthesis of the tricyclic intermediate chanoclavine-I. Disruption of easA , a gene predicted to encode a flavin-dependent oxidoreductase of the old yellow enzyme class, in A. fumigatus led to accumulation of chanoclavine-I and chanoclavine-I-aldehyde. Complementation of the A. fumigatus easA mutant with a wild-type allele from the same fungus restored the wild-type profile of ergot alkaloids. These data demonstrate that the product of A. fumigatus easA is required for incorporation of chanoclavine-I-aldehyde into more-complex ergot alkaloids, presumably by reducing the double bond conjugated to the aldehyde group, thus facilitating ring closure. Augmentation of the A. fumigatus easA mutant with a homologue of easA from Claviceps purpurea resulted in accumulation of ergot alkaloids typical of clavicipitaceous fungi (agroclavine, setoclavine, and its diastereoisomer isosetoclavine). These data indicate that functional differences in the easA -encoded old yellow enzymes of A. fumigatus and C. purpurea result in divergence of their respective ergot alkaloid pathways.

A method for transfructosylation of ergot alkaloids elymoclavine, chanoclavine, lysergol, 9, 10-dihydrolysergol using commercial yeast β-fructofuranosidase and sucrose is described

Clavine alkaloid production in submerged cultures of various Claviceps strains was inhibited by 0.25 mM 5-fluorotryptophan (5-Ftrp). The inhibition was removed by washing the cultures, or by addition of 50 mM tryptophan. Lower doses of tryptophan (0.25–25.0 mM) were ineffective. Addition of cycloheximide and/or 8-hydroxyquinoline (8-HOQ) to be inhibited cultures 24 and 48 h prior washing increased the rate of alkaloid synthesis in washed cultures (superinduction) and the rate of clavine synthesis was proportional to the length of 8-HOQ pretreatment. The addition of 0.25 mM tryptophan in washed cultures also stimulated alkaloid synthesis, but only for 2–3 days, then production in the cultures without tryptophan was higher. Cultures of Claviceps strains 59 and EK-10 inhibited with 5-Ftrp were capable of converting chanoclavine into further clavine pathway products. 5-Fluorotryptophan probably acts as an inhibitor of the first enzyme of the clavine pathway. However, the superinduction suggests the presence of a labile regulatory protein controlling clavine biosynthesis.

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-β-d-fructofuranosyl(2→1)-O-β-d-fructofuranoside, and elymoclavine-O-β-d-fructoside, the respective fructosides of chanoclavine, lysergol and dihydro-lysergol monofructosides were obtained. These substances are formed by β-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.

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus , a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea . We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW , the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus , and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea , as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.

金沢大学大学院自然科学研究科生理活性物質科学, 金沢大学薬学部

Ergot alkaloids are mycotoxins produced mainly by the genera Claviceps, Aspergillus and Penicillium. Natural and semisynthetic ergot alkaloids are used as drugs in modern medicine. Chanoclavine-I aldehyde was found to be the branch point in the biosynthesis of different ergot alkaloid types. In Claviceps purpurea, it was converted to agroclavine by agroclavine synthase EasG in the presence of reduced glutathione, while festuclavine synthase FgaFS and the old yellow enzyme FgaOx3 from Aspergillus fumigatus catalysed the formation of festuclavine by two reduction steps. In this study, pyroclavine was detected as a minor product in the incubation mixture with enzymes from A. fumigatus under optimised HPLC conditions. Furthermore, we demonstrated that the homologues of FgaFS and FgaOx3 from Penicillium commune, FgaFSpc and FgaOx3pc, also catalysed the formation of pyroclavine and festuclavine, but with a significantly higher yield for pyroclavine. In addition, EasG was also shown to convert chanoclavine-I aldehyde to pyroclavine and festuclavine in the presence of the old yellow enzyme FgaOx3 or FgaOx3pc. More importantly, the ratio of pyroclavine to festuclavine was controlled by the reduction of an imine intermediate catalysed by FgaFS, FgaFSpc or EasG, which was proven by using different combinations of these enzymes in the presence of FgaOx3 or FgaOx3pc. The structure of pyroclavine was unequivocally elucidated by NMR and MS analyses.

Four species of Balansia (clavicipitaceous systemic grass pathogens) isolated from pastures where cattle showed signs of ergot toxicity were grown in culture. Balansia epichloe, 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 transferred 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 epichloe 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. epichloe, indicating that this endophyte produces alkaloids both in vivo and in vitro.

The conversions of clavine-type alkaloids have been studied by the use of non-labeled alkaloids and the acetone-treated mycelia of the strains of the Claviceps as well as those of the Aspergillus, Penicillium and others which were productive of clavine-type ergot alkaloids. It was found that agroclavine could be converted to elymoclavine not only by ergot fungi but also by the fungus other than ergot fungi. This finding indicates that the theory presented by authors that elymoclavine is the primary product of the ergot alkaloids has become untenable to some extent. While, it was found that, besides the previous cyclic route, elymoclavine→lysergolI→lysergene→agroclavine→elymoclavine, another biosynthetic route from agroclavine to elymoclavine through lysergene, lysergol and an unestablished alkaloid PS might exist in various fungi on the one hand. This finding indicates that agroclavine rather than elymoclavine will be the primary product of the alkaloids, and that chanoclavine (secaclavine) will be a biosynthetic precursor of agroclavine, and therefore, of the remaining alkaloids. Judging from the data of Stauffacher et al., it was presumed that the reversible reaction, chanoclavine_??_agroclavine, which was found in all of the tested strains would be effected through a certain intermediary compound, possibly iso-chanoclavine-(I), and indeed this intermediary compound would be an obligatory precursor of agroclavine. The present finding indicates that the conversion reaction, elymoclavine→peptide-type alkaloids, cannot be effected through lysergol, but possibly through 6-methyl-Δ8, 9-ergolene-8-carboxylic acid which was discovered by Kobel et al. The outline of this study was given at the 3 rd International Symposium on “Biochemistry and Physiology of Alkaloids” held in Halle (Saale), June 24_??_27, 1965.

The reaction sequence tryptophan →chanoclavine-I → agroclavine → elymoclavine →lysergic acid derivatives was demonstrated in Claviceps. Incorporation of chanoclavine-I into ergoline derivatives ranged up to 40 per cent. No conversion of elymoclavine or agroclavine into chanoclavine-I could be demonstrated.

Using a strain of Claviceps purpurea which produces under submerged conditions chanoclavine, ergometrine and mainly ergotoxine the biosynthesis of ergoline derivatives has been studied. DL-Tryptophan- (ring-2-14C) is specifically incorporated into the lysergic acid moiety of ergotoxine· alkaloids. L-Proline-U-14C serves as a precursor of the proline-part of the peptid-chain of ergotoxine.

Psychotomimetic substances in cultivated morning-glory seed have resulted in a prohibition of morning-glory seed in soybean sold internationally. Seeds of four native, wild morning glory species-purple moon-flower (Calonyction muricatum), smallflower morning glory (Jacquemontia tamnifolia), cypressvine (Quamoclit lobata), and cardinal creeper (Q. sloteri)-were analyzed for ergot alkaloid contents. Seed of a horticultural variety of Ipomoea violaceae `Heavenly Blue' contained 0.052% total alkaloids. Total ergot alkaloids in the wild morning-glory species ranged from 0.0029% to 0.0059% (5.6%-11.4% of the total alkaloids in `Heavenly Blue' seed). Major alkaloids were separated and characterized by two-dimensional thin-layer chromatography and co-chromatography with authenticated standards as chanoclavine, elymoclavine, penniclavine, ergonovinine, agroclavine, ergonovine, ergosine, and ergosinine. The types of alkaloids varied among morning-glory species, and the quantities were too small to be considered a p...

The simple total synthesis of the ergot alkaloids, (±)-norchanoclavine-I, (±)-chanoclavine-I, (±)-isochanoclavine-I, and (±)-agroclavine was achieved by a practical and common synthesis method. A new regio-selective oxidation of the Z-methyl group of the isoprenyl system with selenium dioxide is described.

Background Ergopeptines are a predominant class of ergot alkaloids produced by tall fescue grass endophyte Neotyphodium coenophialum or cereal pathogen Claviceps purpurea. The vasoconstrictive activity of ergopeptines makes them toxic for mammals, and they can be a problem in animal husbandry. Results We isolated an ergopeptine degrading bacterial strain, MTHt3, and classified it, based on its 16S rDNA sequence, as a strain of Rhodococcus erythropolis (Nocardiaceae, Actinobacteria). For strain isolation, mixed microbial cultures were obtained from artificially ergot alkaloid-enriched soil, and provided with the ergopeptine ergotamine in mineral medium for enrichment. Individual colonies derived from such mixed cultures were screened for ergotamine degradation by high performance liquid chromatography and fluorescence detection. R. erythropolis MTHt3 converted ergotamine to ergine (lysergic acid amide) and further to lysergic acid, which accumulated as an end product. No other tested R. erythropolis strain degraded ergotamine. R. erythropolis MTHt3 degraded all ergopeptines found in an ergot extract, namely ergotamine, ergovaline, ergocristine, ergocryptine, ergocornine, and ergosine, but the simpler lysergic acid derivatives agroclavine, chanoclavine, and ergometrine were not degraded. Temperature and pH dependence of ergotamine and ergine bioconversion activity was different for the two reactions. Conclusions Degradation of ergopeptines to ergine is a previously unknown microbial reaction. The reaction end product, lysergic acid, has no or much lower vasoconstrictive activity than ergopeptines. If the genes encoding enzymes for ergopeptine catabolism can be cloned and expressed in recombinant hosts, application of ergopeptine and ergine degrading enzymes for reduction of toxicity of ergot alkaloid-contaminated animal feed may be feasible.

An alkaloid-blocked mutant ofClaviceps purpurea was isolated from a strain which produces ergotoxine alkaloids. The mutant accumulates chanoclavine-I and the corresponding aldehyde. It lacks the ability to form tetracyclic ergolines.

OVER the past few years we have been examining the composition of ergot alkaloids produced in vitro by strains of Claviceps purpurea isolated from sclerotia growing on grasses in England. It was noted that exhaustive chloroform extraction of culture filtrates adjusted to pH 8.5 removed only about 40 per cent of the total Ehrlich-positive material; the remaining material at pH 3.5 was readily extracted with n-butanol. Paper1,2 and thin-layer3 chromatographic analysis of the chloroform-soluble fraction showed chanoclavine to be the principal component present accompanied by small quantities of elymoclavine and the ergotoxine group. Chromatography of the butanol fraction revealed only one fluorescent, Ehrlich-positive spot which was inseparable from D-lysergic acid and 6-methylergol-8-ene-8-carboxylic acid, the isomer of lysergic acid recently obtained from cultures of C. paspali4. We now have re-examined culture filtrates of a strain of C. purpurea originally isolated from Spartina townsendii to investigate further the composition of the butanol fraction.