Biotechnology and genetics of ergot alkaloids

Ergot alkaloids, i.e. ergoline-derived toxic metabolites, are produced by a wide range of fungi, predominantly by members of the grass-parasitizing family of the Clavicipitaceae. Naturally occurring alkaloids like the D-lysergic acid amides, produced by the "ergot fungus" Claviceps purpurea, have been used as medicinal agents for a long time. The pharmacological effects of the various ergot alkaloids and their derivatives are due to the structural similarity of the tetracyclic ring system to neurotransmitters such as noradrenaline, dopamine or serotonin. In addition to "classical" indications, e.g. migraine or blood pressure regulation, there is a wide spectrum of potential new applications of this interesting group of compounds. The biotechnology of ergot alkaloids has a long tradition, and efficient parasitic and submerse production processes have been developed; the biochemistry of the pathway and the physiology of production have been worked out in detail. The recent identification of a cluster of genes involved in ergot alkaloid biosynthesis in C. purpurea and the availability of molecular genetic techniques allow the development of strategies for rational drug design of ergoline-related drugs by enzyme engineering and by biocombinatorial approaches.     

Derivation of the multiply-branched ergot alkaloid pathway of fungi

Ergot alkaloids are a large family of fungal specialized metabolites that are important as toxins in agriculture and as the foundation of powerful pharmaceuticals. Fungi from several lineages and diverse ecological niches produce ergot alkaloids from at least one of several branches of the ergot alkaloid pathway. The biochemical and genetic bases for the different branches have been established and are summarized briefly herein. Several pathway branches overlap among fungal lineages and ecological niches, indicating activities of ergot alkaloids benefit fungi in different environments and conditions. Understanding the functions of the multiple genes in each branch of the pathway allows researchers to parse the abundant genomic sequence data available in public databases in order to assess the ergot alkaloid biosynthesis capacity of previously unexplored fungi. Moreover, the characterization of the genes involved in the various branches provides opportunities and resources for the biotechnological manipulation of ergot alkaloids for experimentation and pharmaceutical development.     

Development of a UHPLC-FLD method for the analysis of ergot alkaloids and application to different types of cereals from Luxembourg

Ergot alkaloids are toxins produced by some species of fungi in the genus Claviceps, that may infect rye and triticale and, in a minor degree, other types of cereals. In this study, a new UHPLC-FLD method for the quantification of the six major ergot alkaloids as well as their corresponding epimers was developed. The sample preparation was done by a solid-liquid extraction with acetonitrile and clean-up via freeze-out. The method was fully validated and then applied to 39 samples (wheat, rye, triticale, and barley) harvested in Luxembourg in 2016. Samples were sieved (1.9?×?20 mm) prior to analysis in order to remove sclerotia, hosting the alkaloids. However, 23 samples still contained at least one ergot alkaloid >?LOQ and concentrations of the sum of the 6 ergot alkaloids ranged from 0.3 to 2530.1 μg/kg. Interestingly, the highest concentrations were measured in wheat and not in rye or triticale, suggesting that all kinds of cereals should be included in monitoring programs. The outcome of this study allowed giving a first overview of ergot alkaloid concentrations in cereals harvested in Luxembourg, and the measured concentrations were in similar ranges than in other parts of the world (e.g., Canada, France, Germany).     

A Complex Ergovaline Gene Cluster in Epichloë Endophytes of Grasses

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.     

Parasitic fungus Claviceps as a source for biotechnological production of ergot alkaloids

Ergot alkaloids produced by the fungus Claviceps parasitizing on cereals, include three major groups: clavine alkaloids, d-lysergic acid and its derivatives and ergopeptines. These alkaloids are important substances for the pharmatech industry, where they are used for production of anti-migraine drugs, uterotonics, prolactin inhibitors, anti-Parkinson agents, etc. Production of ergot alkaloids is based either on traditional field cultivation of ergot-infected rye or on submerged cultures of the fungus in industrial fermentation plants. In 2010, the total production of these alkaloids in the world was about 20,000 kg, of which field cultivation contributed about 50%. This review covers the recent advances in understanding of the genetics and regulation of biosynthesis of ergot alkaloids, focusing on possible applications of the new knowledge to improve the production yield.     

An Ergot Alkaloid Biosynthesis Gene and Clustered Hypothetical Genes from Aspergillus fumigatus

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.     

A complex ergovaline gene cluster in epichloe endophytes of grasses

Clavicipitaceous fungal endophytes of the genera Epichlo? and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.     

Distribution of ergot alkaloids and ricinoleic acid in different milling fractions

The sclerotia of the fungus Claviceps sp. are still a challenge for the milling industry. Ergot sclerotia are a constant contamination of the rye crop and have to be removed by modern milling technologies. Changing sizes and coloration of the sclerotia make it difficult to separate them from the grain. Ergot sclerotia are a problem when cleaning is insufficient and non-separated specimens or sclerotia fragments get into the milling stream and thus ergot alkaloids are distributed into the different cereal fractions. In model milling experiments, the residues of ergot in rye flour and the distribution of ergot into different milling fractions were investigated. Rye grains were mixed with whole ergot sclerotia and in another experiment with ergot powder and cleaned afterwards before milling. The ergot alkaloids ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristineand their related isomeric forms (-inine-forms), and additionally ricinoleic acid as a characteristic component of ergot, were quantified in the different milling fractions. From the first experiment, it can be shown that after harvesting even simple contact of sclerotia with bulk grains during ordinary handling or movement of bulk grain in the granary is sufficient to contaminate all the healthy or sound rye grains with ergot alkaloids. Thereby, the amount of ergot residue correlates with the amount of peripheral layers of rye grains in the flour. In an additional experiment without sclerotia specimens, bulk rye grains were loaded with powder of sclerotia. After subsequent cleaning, aconcentration of ergot alkaloids was detected, which was tenfold higher than the ergot alkaloidconcentration of the experiment with intact ergot sclerotia.     

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.     

An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus

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.     

An Old Yellow Enzyme Gene Controls the Branch Point between Aspergillus fumigatus and Claviceps purpurea Ergot Alkaloid Pathways

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.Different classes of ergot alkaloids are produced by members of two distinct fungal lineages. Clavicipitaceous species, which include Claviceps spp. and Neotyphodium spp., are in the order Hypocreales and typically synthesize lysergic acid derivatives (13, 16, 18). These alkaloids have a double bond in the last assembled of four rings (D ring) of the tetracyclic ergoline ring structure. Ergot alkaloids are also produced by the distantly related opportunistic human pathogen Aspergillus fumigatus, a member of the order Eurotiales (8, 14, 16, 18). Ergot alkaloids of A. fumigatus are of the clavine class and differ from the more complex profile of Claviceps purpurea and Neotyphodium spp. One important distinction between the ergot alkaloids produced by these different fungi is the saturation of the fourth ring of the ergoline structure in A. fumigatus (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Structures and relationships of relevant ergot alkaloids. (A) Chanoclavine-I is oxidized to its aldehyde form before being incorporated into festuclavine (and downstream alkaloids) in A. fumigatus or agroclavine (and downstream alkaloids) in C. purpurea. (B) Conventional ring labeling and atom numbering referred to in the text.Several genes involved in the ergot alkaloid pathways of A. fumigatus and clavicipitaceous fungi are found clustered together in the genome of each species (3, 4, 6, 18, 23). These distantly related fungi are hypothesized to share several early pathway steps, after which the pathways diverge to yield distinct sets of ergot alkaloids (3, 13, 16). The gene dmaW, which encodes dimethylallyltryptophan (DMAT) synthase, catalyzes the prenylation of tryptophan that initiates the ergot alkaloid pathway in clavicipitaceous fungi (22, 25) and functions similarly in A. fumigatus (3, 24). The region surrounding this gene in A. fumigatus contains homologues of genes also found in Neotyphodium lolii and C. purpurea ergot alkaloid gene clusters (3, 4, 6). One of the shared genes, easA, is predicted to encode a member of the old yellow enzyme (OYE) family of oxidoreductases. Old yellow enzymes are flavin-containing oxidoreductases initially found in the brewer''s bottom yeast Saccharomyces carlsbergensis (26). Enzymes in this family use a reduced flavin cofactor and an active-site tyrosine residue to reduce the carbon-carbon double bond in an α/β-unsaturated aldehyde or ketone (7, 10). Subsequently, the enzymes require NADPH to restore the flavin cofactor to its reduced state. OYEs catalyze multiple reactions useful for both biotechnological and pharmaceutical applications; however, physiological roles and natural substrates for many of these enzymes presently are unknown (26). On the basis of the apparent need in the ergot alkaloid pathway of A. fumigatus for reduction of a carbon-carbon double bond in the intermediate chanoclavine-I-aldehyde, we hypothesized that the OYE-encoding gene easA is required for ergot alkaloid biosynthesis (3, 16). In this study, easA in A. fumigatus was disrupted and complemented to ascertain the role of its gene product in ergot alkaloid biosynthesis.     

Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the Polymerase Chain Reaction

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the generaPenicilliumand Clavicepswas evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerate primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as -cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway. Received: 26 August 1998 / Accepted: 19 October 1998     

Biodiversity of Convolvulaceous species that contain ergot alkaloids,indole diterpene alkaloids,and swainsonine

Convolvulaceous species have been reported to contain several bioactive principles thought to be toxic to livestock including the calystegines, swainsonine, ergot alkaloids, and indole diterpene alkaloids. Swainsonine, ergot alkaloids, and indole diterpene alkaloids are produced by seed transmitted fungal symbionts associated with their respective plant host, while the calystegines are produced by the plant. To date, Ipomoea asarifolia and Ipomoea muelleri represent the only Ipomoea species and members of the Convolvulaceae known to contain indole diterpene alkaloids, however several other Convolvulaceous species are reported to contain ergot alkaloids. To further explore the biodiversity of species that may contain indole diterpenes, we analyzed several Convolvulaceous species (n = 30) for indole diterpene alkaloids, representing four genera, Argyreia, Ipomoea, Stictocardia, and Turbina, that had been previously reported to contain ergot alkaloids. These species were also verified to contain ergot alkaloids and subsequently analyzed for swainsonine. Ergot alkaloids were detected in 18 species representing all four genera screened, indole diterpenes were detected in two Argyreia species and eight Ipomoea species of the 18 that contained ergot alkaloids, and swainsonine was detected in two Ipomoea species. The data suggest a strong association exists between the relationship of the Periglandula species associated with each host and the occurrence of the ergot alkaloids and/or the indole diterpenes reported here. Likewise there appears to be an association between the occurrence of the respective bioactive principle and the genetic relatedness of the respective host plant species.     

Zum Einfluss einer hydrothermischen Behandlung auf den Ergotalkaloidgehalt von mutterkornbelastetem Roggen

Hydrothermal treatments are primarily used to increase the digestibility of nutrients and therefore to improve the feeding value of feedstuffs mainly for non-ruminants. Other positive side effects may occur, e.g. a decrease in toxicity of feed contaminated with mycotoxins. To study such effects, 4 batches of rye containing different percentages (0.8, 4.2, 8.3 and 25%) of ergot (Claviceps purpurea) were expanded and ergot alkaloid contents were analysed. After pre-conditioning of each batch by steam exposure for approx. 2 min, at 95 °C and 17% moisture, the material was expanded for approx. 5 sec. at 120 °C, 18% moisture, 40 bar mechanical pressure and 20 kWh/t mechanical energy input. Samples were collected before and after pre-conditioning and after expanding. Ergot alkaloids were analysed by HPLC. Analysis includedErgometrine, Ergotamine, Ergocornine, Ergocryptine, Ergocristine, Ergosine and their respective-inine isomers, the sum of these 12 ergot alkaloids was referred to as the total alkaloid content. On average, the hydrothermal treatment (pre-conditioning and expanding) caused a decrease of the total ergot alkaloid content of approx. 10%. Except for the batch containing 0.8% ergot, the efficiency of the hydrothermal treatment decreased with increasing ergot concentration in the batches. In general, the hydrothermal treatment changed the proportions of the ergot alkaloid isomers since the percentages of the-inine isomers of the total ergot alkaloid contents were increased with reduced-ine percentages. Whether this alteration is of toxicological relevance should be evaluated in animal experiments.     

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.     

Physiological status of submergedClaviceps during enzymic assembly of ergot alkaloids

Ergot alkaloids are formed only for arelatively brief time during the lifespan of the culture and under conditions of reduced proliferation. They cannot be taken for waste products of general metabolism. Ergot strains are capable of carrying out all the simple steps of the anthranilic acid—tryptophan cycles. Alkaloids influence activities of certain enzymes of primary metabolism in the ergot mycelium,e.g. tryptophan synthetase, acetyl-CoA carboxylase, citrate synthase, isocitrate lyase, and malate synthase. Ergot alkaloids do not belong to a group of physiologically inert secondary metabolites. A tentative scheme of the enzymic assembly of the ergoline nucleus is presented. The increased yield of ergoline alkaloids may be attributed to the following points: (1) Unbalnced growth of the culture. (2) Support of competition of fatty acids and alkaloid biosynthesis for acetyl-CoA. (3) Decreased activities of tricarboxylic acid and glyoxylate cycles. (4) Positive association between the rate of protein turnover and alkaloid formation. (5) Stimulation of both tryptophan synthesis and degradation via kynurenine—anthranilate. (6) Regulation of tryptophan-histidine cross-pathway. (7) Continuous control of the alkaloid level during fermentation.     

Association of ergot alkaloids with conidiation in Aspergillus fumigatus

Ergot alkaloids are mycotoxins that affect the nervous and reproductive systems of exposed individuals through interactions with monoamine receptors. They have been studied more widely in ergot fungi and grass endophytes but also are found in Aspergillus fumigatus, an opportunistic human pathogen that reproduces and disseminates exclusively through conidia. The ergot alkaloids festucla-vine and fumigaclavines A, B and C are present in or on conidia of A. fumigatus. Cultures of the fungus that are free of conidia are difficult to obtain, obscuring comparisons of conidia versus vegetative hyphae as sources of the ergot alkaloids. To create conidiation-deficient strains of A. fumigatus we manipulated the bristle A gene (brlA), which controls vesicle formation or budding growth necessary for conidiation in Aspergillus spp. Disruption of brlA in A. fumigatus, via homologous recombination, resulted in a nonconidiating mutant that produced bristle-like structures instead of conidiophores and conidia. Moreover the disrupted strain failed to produce ergot alkaloids as verified by HPLC analyses. Complementation with a wild-type allele restored conidiation and ergot alkaloid production. These results suggest that ergot alkaloids are not produced within the vegetative mycelium of the fungus and are associated directly with conidiation.     

The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., H?lter, 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 68.5kb) 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.     

Chemotypic diversity of epichloae,fungal symbionts of grasses

The epichloid fungi – comprising sexual Epichloë species and asexual Neotyphodium species – are symbionts of cool-season grasses (subfamily Poöideae), mostly vertically transmissible (seedborne), and well known for production of anti-herbivore alkaloids. Four classes of alkaloids are known to be produced by epichloae: lolines (saturated aminopyrrolizidines), indole–diterpenes, ergot alkaloids, and peramine. There is a wide range of chemotypic diversity among and even within epichloid species. At the molecular level, this diversity may in part reflect the telomeric association of two of the four alkaloid biosynthesis gene clusters. Ecologically, the chemotypic diversity within species may reflect frequency-dependent selection for the alkaloids, which provide defences against insects and, in some cases, vertebrates, but can be expensive to produce. Interspecific hybridization, common among asexual epichloae, can pyramid the alkaloid biosynthesis genes. Compared to sexual epichloae, many asexual epichloae produce high levels of alkaloids – particularly lolines – suggesting that strict vertical transmission selects for enhanced capability of host protection.