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Four ergot species (Claviceps ripicola, C. quebecensis, C. perihumidiphila, and C. occidentalis) were recognized based on analyses of DNA sequences from multiple loci, including two housekeeping genes, RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α), and a single-copy ergot alkaloid synthesis gene (easE) encoding chanoclavine I synthase oxidoreductase. Morphological features, ergot alkaloid production, and pathogenicity on five common cereal crops of each species were evaluated and presented in taxonomic descriptions. A synoptic key was also provided for identification.

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.

Ergot alkaloids belong to a diverse group of fungal-derived natural compounds with a range of biological activities that have important applications in medicine and agriculture. Cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety, which possesses a very broad insecticidal activity, was produced in high levels in Saccharomyces cerevisiae. It is the first ergot alkaloid downstream of chanoclavine to be successfully synthesized in a heterologous host in excellent yields. The insecticidal activity of the natural product itself was not sufficient for commercial use, but the broad activity in combination with the insect-specific mode of action motivated us to initiate a chemistry program. First, we undertook the modification of cycloclavine itself, followed by synthesis of simpler analogs in order to find compounds with enhanced insecticidal activity.

While the use of ergot alkaloids in folk medicine has been practiced for millennia, systematic investigations on their therapeutic potential began about 100 years ago. Subsequently, Albert Hofmann's discovery of lysergic acid diethylamide (LSD) and its intense psychedelic properties garnered worldwide attention and prompted further studies of this compound class. As a result, several natural ergot alkaloids were discovered and unnatural analogs were synthesized, and some were used to treat an array of maladies, including Alzheimer's and Parkinson's disease. While LSD was never commercially approved, recent clinical studies have found it can be an innovative and effective treatment option for several psychiatric disorders. Ongoing biosynthetic and total synthetic investigations aim to understand the natural origins of ergot alkaloids, help develop facile means to produce these natural products and enable their continued use as medicinal chemistry lead structures. This review recounts major developments over the past 20 years in biosynthetic, total synthetic, and pharmaceutical studies. Many ergot alkaloid biosynthetic pathways have been elucidated, with some of them subsequently applied toward "green" syntheses. New chemical methodologies have fostered a fast and efficient access to the ergoline scaffold, prompting some groups to investigate biological properties of natural product-like ergot alkaloids. Limited pharmaceutical applications have yet to completely bypass the undesirable side effects of ergotism, suggesting further studies of this drug class are likely needed and will potentially harness major therapeutic significance.

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.

Biomimetic total syntheses of either enantiomers of a number of ergot alkaloids, chanoclavine I (1b), chanoclavine I aldehyde (1c), pyroclavine (1e), festuclavine (1f), pibocin A (1g), 9-deacetoxyfumigaclavine C (1h), and fumigaclavine G (1i), have been achieved from seco-agroclavine (1a). The advanced intermediate for seco-agroclavine (1a) was synthesized via a key thiourea-catalyzed intramolecular nitronate addition onto α,β-unsaturated ester.

The blue cheese-making fungus Penicillium roqueforti produces isofumigaclavine A as the main ergot alkaloid. Recently, genome mining revealed the presence of two DNA loci bearing the genetic potential for its biosynthesis. In this study, a short-chain dehydrogenase/reductase (SDR) from one of the loci was proved to be responsible for the conversion of chanoclavine-I to its aldehyde. Furthermore, a putative gene coding for an enzyme with high homology to Old Yellow Enzymes (OYEs) involved in the ergot alkaloid biosynthesis was found outside the two clusters. Biochemical characterisation of this enzyme, named FgaOx3Pr3, showed that it can indeed catalyse the formation of festuclavine in the presence of a festuclavine synthase FgaFS, as had been observed for other OYEs in ergot alkaloid biosynthesis. Differing from other homologues, FgaOx3Pr3 does not convert chanoclavine-I aldehyde to its shunt products in the absence of FgaFS. Instead, it increases significantly the product yields of several SDRs for the conversion of chanoclavine-I to its aldehyde. Kinetic studies proved that overcoming the product inhibition is responsible for the observed enhancement. To the best of our knowledge, this is the first report on the bifunctionality of an OYE and its synergistic effect with SDRs.

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.

A rapid, simple, sensitive, gradient and reproducible, reverse-phase high-performance liquid chromatographic method was developed for the quantitative estimation of bioactive alkaloids, lysergol and chanoclavine in the seeds of Ipomoea muricata. The clavine alkaloid, lysergol, is a bioenhancer for the drugs and nutrients. The samples were analyzed by reverse-phase chromatography on a Waters spherisorb ODS2 column (250 × 4.6 mm, i.d., 10 µm) using binary gradient elution with acetonitrile and 0.01 m phosphate buffer (NaH2PO4) containing 0.1% glacial acetic acid at a flow rate of 0.8 mL/min, a column temperature of 25 °C and UV detection at λ 254 nm. The limits of detection (LOD) and quantitation (LOQ) were 0.035 and 0.106 µg/mL for lysergol and 0.039 and 0.118 µg/mL for chanoclavine, respectively. Standard curves were linear in the range of 2–10 µg/mL (r > 99) for both analytes. Good results were achieved with respect to repeatability (RSD

A previous study showed that together with the festuclavine synthase FgaFS, the old yellow enzyme FgaOx3 from Aspergillus fumigatus catalyzed the conversion of chanoclavine-I aldehyde to festuclavine in the biosynthesis of ergot alkaloids. In the absence of FgaFS, a mixture containing two compounds with a ratio of 7:3 was detected in the enzyme assay of FgaOx3. NMR experiments including (DQF)-COSY, HSQC, HMBC and NOESY identified their structures as E/Z isomers of N-methyl-N-[(5R,10R)-10-(2-oxo-propyl)-2,4,5,10-tetrahydrobenzo[cd]indol-5-yl]formamide and proved the migration of the formyl group at C-8 in chanoclavine I-aldehyde to N-6 in the identified products. Copyright © 2011 John Wiley & Sons, Ltd.

This account describes the development of a Diels-­Alder-based methodology towards highly functionalized dihydronaphthalenamines, and their conversion into indoles through a modified Plieninger procedure. Applications towards the total synthesis of indole-containing natural products will be presented. 1 Introduction 2 Synthesis of Indoles via Diels-Alder Cycloadditions/­Plieninger Indolization 2.1 High Pressure Diels-Alder Cycloadditions of Quinone ­Imine Ketals (QIKs) 2.2 Thermal Diels-Alder Cycloadditions of QIKs 2.3 Development of a Modified Plieninger Protocol 2.4 Diels-Alder Cycloadditions/Indolization of p-Benzoquin­one Monoimines 3 Applications to Natural Products Synthesis 3.1 Ergot Alkaloids: Chanoclavine I (Plieninger) 3.2 Rivularins (Maehr) 3.3 Polyalkylated Indoles: Herbindoles and Trikentrins (Kerr) 3.4 Tremorgenic Indole Terpenoids: Lolicine Western Hemisphere (Kerr) 3.5 Antitumor Agents: Yatakemycin (Boger) and CC-1065 (Kraus, Kerr) 3.6 Antimalarials: Decursivine (Kerr) 3.7 Carbazole Natural Products: The Clausamines and Eustifolines (Kerr) 4 Summary and Outlook

An existing LC–MS/MS method for multi-mycotoxin determination was extended by further 19 analytes and was applied for a semi-quantitative screening of 87 mouldy food samples from private households, including bread, fruits, vegetables, cheeses, nuts and jam. In the 247 investigated sub-samples, 49 different analytes were identified, some of which were never reported before to occur in naturally contaminated food. Enniatins and ergot alkaloids occurred in all samples of (dark) bread/pastries at low μg/kg-levels. From the remaining analytes, chanoclavine, emodin, mycophenolic acid and roquefortine C were found most frequently. Regulated mycotoxins occurred less often, but the corresponding concentrations exceeded the regulatory limits up to a factor of 1000 in case of patulin. Moreover, considerable mycotoxin concentrations were observed in some sub-samples taken from non-mouldy spots of the investigated samples. Thus, it was concluded that it is not safe to remove the mouldy part and consume the remainder.

Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His6-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD+ resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K M values for chanoclavine-I and NAD+ were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s−1.

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids, directly from a crude extract of Ipomoea muricata. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)–acetonitrile–water (4:1:5, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and trifluoroacetic acid (10 mM) to the aqueous mobile phase as an eluter. From 4 g of crude extract, 210 mg lysergol and 182 mg chanoclavine were obtained in 97% and 79.6% purities. Total yield recovery was >95%. Isolated alkaloids were characterized on the basis of their 1H, 13C NMR and ESI-MS data.

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.

Clavines and D-lysergic acid-derived alkaloid amides and alkaloid peptides are two different families of compounds that have the indole-derived tetracyclic metergoline ring system in common. Previous work has shown that D-lysergic acid is biosynthetically derived from clavine alkaloids. Recent cloning and analysis of the ergot alkaloid biosynthesis gene cluster from the D-lysergic acid peptide (ergopeptines)-producing Claviceps purpurea, has shown that it most probably contains all genes necessary for D-lysergic acid synthesis as well as those that encode the assembly of D-lysergic acid peptides, such as ergotamine. To address the role of the oxygenase genes of alkaloid-gene clusters, the only cytochrome P450 monooxygenase gene of this cluster was inactivated by disruption. The resultant mutant accumulated agroclavine, elymoclavine, and chanoclavine in substantial amounts but not ergopeptines. Feeding the mutant with D-lysergic acid restored ergopeptine synthesis; this suggests a block in the conversion of elymoclavine to D-lysergic acid. The gene was designated cloA (for encoding a clavine oxidase, CLOA). Retransformation of the mutant with the intact cloA gene also restored ergopeptine synthesis. These data show that CLOA catalyses the conversion of clavines to D-lysergic acid, it acts as a critical enzyme in the ergot alkaloid gene cluster, and bridges the biosynthesis of the two different families of alkaloids.

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., Holter, K., Correia, T., Arntz, C., Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133–141] was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5 kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.

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.

The mutualistic symbiosis following infection of tall fescue, Festuca arundinacea, and perennial ryegrass, Lolium perenne, by fungal endophyte (Neotyphodium spp.) results in the production of alkaloids that are feeding deterrents or toxic to insects and livestock. If the levels of the alkaloids can be manipulated by cultural practices in the grasses that are used for home lawns and golf courses, this could alleviate the need for pesticide applications in urban environments. We evaluated the influence of mowing height on the levels of some alkaloids in a greenhouse experiment for two consecutive months. In tall fescue, levels of four of the nine alkaloids, including one presumptive alkaloid, showed increased levels with increasing the mowing height from 2.5 to 7.5 cm. The alkaloids were ergonovine, ergocryptine, perloline methyl ether, and an unidentified alkaloid designated as unknown C. In perennial ryegrass, three out of six alkaloids, perloline methyl ether, chanoclavine, and unknown A, showed similar increases. The alkaloid levels in perennial ryegrass showed more variability than those in tall fescue between the two sampling dates. It was clear in both grasses that the relative levels of the alkaloids varied with mowing height, as well as over time.

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.

New isomers of clavine alkaloids with distinctively low chromatographic mobilities were isolated from the collection and mutant strains of Penicillium roquefortiiThom 1906, in addition to alkaloids roquefortine, 3,12-dihydroroquefortine, isofumigaclavines A and B, festuclavine, and chanoclavine-I, which are characteristic of this species. It was demonstrated that the collection strain produces isomers of agroclavine and epoxyagroclavine, whereas the mutant strain synthesizes isomers of fumigaclavines A and B, festuclavine, and chanoclavine.

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 content of ergot alkaloids (ergovaline and chanoclavine), and their production in October 1996 and during the period May–September 1997, were investigated in seventeen ecotypes of perennial ryegrass (Lolium perenne L.) and in nineteen ecotypes of meadow fescue (Festuca pratensis Huds.), naturally infected with Neotyphodium spp. The ecotypes were collected in the north-eastern part of the Czech Republic. In 1996 the content of ergovaline in the ecotypes of perennial ryegrass ranged from 0·00 to 2·73 μg g–1 dry matter (DM) (one cut), and in 1997 from 0·00 to 4·65 μg g–1 DM (five cuts). In meadow fescue the content of ergovaline varied from 0·00 to 0·61 μg g–1 DM (one cut) in 1996, and in 1997 from 0·00 to 2·31 μg g–1 DM (five cuts). The content of chanoclavine (investigated in 1997 in four cuts only) in perennial ryegrass ranged between 0·00 and 3·39 μg g–1 DM, and in meadow fescue between 0·00 and 2·26 μg g–1 DM. Most ecotypes of L. perenne reacted to the high temperature and heavy rainfall in June and July of 1997 with an enhanced production of ergovaline, whereas the content of chanoclavine was not changed. Such reaction to stress conditions was not observed in the ecotypes of F. pratensis. Large differences in the production of both ergot alkaloids between different ecotypes of both plant species were observed.

The total synthesis of optically active clianochvine-I, an ergot alkaloid, was accomplished using palladium-catalyzed intramoleeular cyclization (Heck reaction) as a key step. The conjugate ester (6) was obtained in 2 steps from optically active 4-bmmotryptophan (10), and the cyclization of 6 proceeded smoothly without racemization to give the key intermediate, trieyelie tetrahydrobenz(c,d)indole derivative (7), in high yield. Copyright © 1996 Elsevier Science Ltd

Structures of oxepino[5,4,3c,d]indole derivatives (2, 3) and 3,4-disubstituted indoles (4, 5) resulting from biotransformation of chanoclavine by Euphorbia calyptrata cell culture have been elucidated by NMR and mass spectroscopy.

Research on livestock toxicoses caused by Acremonium (endophyte)-infected grasses strongly implicate the ergopeptine alkaloids with A. coenophialum-infected fescue and paxilline and the lolitrem alkaloids with A. lolii-infected perennial ryegrass as the causative agents. Isolation, identification, and detection of these toxins involves extraction with appropriate solvents, clean-up procedures, and chromatographic methods with known standards. Thin-layer, high-performance liquid and gas chromatography along with ultraviolet and mass spectrometric (i.e., electron impact, chemical ionization, tandem mass) characterizations have been reported. These methods have varying degrees of success depending on the matrix from which the alkaloids have been extracted. Ergovaline is the primary ergopeptine alkaloid isolated from cultures of A. coenophialum and also from infected fescue grass and seeds toxic to livestock. Other compounds isolated from the endophyte-infected fescue include: lysergic acid amide (ergine), the clavine class of ergot alkaloids (chanoclavine I, agroclavine, elymoclavine, penniclavine), the pyrrolizidine alkaloids (N-formylloline, N-acetylloline, N-methyloline, N-acetylnorloline), and the unique pyrrolopyrazine alkaloid peramine. The loline alkaloids and peramine have been more associated with the insect-deterrent properties of the endophyte-infected fescue than with livestock toxicoses. Also, both peramine and the ergopeptine alkaloids (ergovaline, ergotamine) have been isolated from A. lolii-infected perennial ryegrass. More recently, paxilline and lolitrem B have been detected in laboratory cultures of A. coenophialum isolated from tall fescue. The ergot alkaloids in endophyte-infected perennial ryegrass may be more related to decreased animal productivity (weight gains, reproduction problems), whereas the lolitrems cause the staggers syndrome. The detection, isolation, identification, and analyses of these compounds from Acremonium-infected grasses is presented.

The total enantioselective synthesis of (−)-chanoclavine I 2 in twelve steps from indole-4-carboxaldehyde 4 is described The key-step 3 → 6 which involves the formation of the C ring by creation of the C5-C10 bond is catalyzed by a chiral palladium (0) complexes. The chiral ergoline synthon 6 is produced with an excellent diastereo-and enantioselectivity (up to 95% ee).

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 %).

2-Acetamido-2-deoxy-O-β-D-galactopyranosides and 2-acetamido-2-deoxy-O-β-D-glucopyranosides of the representatives of the three classes of the ergot alkaloids, i.e., elymoclavine (1a) (clavines), chanoclavine (2a) (secoclavines) and ergometrine (3a)/ergometrinine (4a) (lysergic/iso-lysergic acid derivatives) were prepared via transglycosylation catalysed by β-N-acetylhexosaminidase from Aspergillus oryzae.

Ergot alkaloids are indole derivatives with diverse structures and biological activities. They are produced by a wide range of fungi with Claviceps purpurea as the most important producer for medical use. Chanoclavine-I aldehyde is proposed as a branch point via festuclavine or pyroclavine to clavine-type alkaloids in Trichocomaceae and via agroclavine to ergoamides and ergopeptines in Clavicipitaceae. Here we report the conversion of chanoclavine-I aldehyde to agroclavine by EasG from Claviceps purpurea, a homologue of the festuclavine synthase FgaFS in Aspergillus fumigatus, in the presence of reduced glutathione and NADPH. EasG comprises 290 amino acids with a molecular mass of about 31.9 kDa. The soluble monomeric His(6)-EasG was purified after overproduction in E. coli by affinity chromatography and used for enzyme assays. The structure of agroclavine was unequivocally elucidated by NMR and MS analyses.

Genes required for ergot alkaloid biosynthesis are clustered in the genomes of several fungi. Several conserved ergot cluster genes have been hypothesized, and in some cases demonstrated, to encode early steps of the pathway shared among fungi that ultimately make different ergot alkaloid end products. The deduced amino acid sequence of one of these conserved genes (easC) indicates a catalase as the product, but a role for a catalase in the ergot alkaloid pathway has not been established. We disrupted easC of Aspergillus fumigatus by homologous recombination with a truncated copy of that gene. The resulting mutant (ΔeasC) failed to produce the ergot alkaloids typically observed in A. fumigatus, including chanoclavine-I, festuclavine, and fumigaclavines B, A, and C. The ΔeasC mutant instead accumulated N-methyl-4-dimethylallyltryptophan (N-Me-DMAT), an intermediate recently shown to accumulate in Claviceps purpurea strains mutated at ccsA (called easE in A. fumigatus) (Lorenz et al. Appl Environ Microbiol 76:1822-1830, 2010). A ΔeasE disruption mutant of A. fumigatus also failed to accumulate chanoclavine-I and downstream ergot alkaloids and, instead, accumulated N-Me-DMAT. Feeding chanoclavine-I to the ΔeasC mutant restored ergot alkaloid production. Complementation of either ΔeasC or ΔeasE mutants with the respective wild-type allele also restored ergot alkaloid production. The easC gene was expressed in Escherichia coli, and the protein product displayed in vitro catalase activity with H(2)O(2) but did not act, in isolation, on N-Me-DMAT as substrate. The data indicate that the products of both easC (catalase) and easE (FAD-dependent oxidoreductase) are required for conversion of N-Me-DMAT to chanoclavine-I.