Ergot Alkaloid Formation Research Articles

Comparative Ergot Alkaloid Elaboration by Selected Plectenchymatic Mycelia of Claviceps purpurea through Sequential Cycles of Axenic Culture and Plant Parasitism.

Ergot alkaloids have an established place in plant pathology and toxicology. As pharmaceuticals, their sourcing is via natural or managed agricultural occurrence of sclerotia of Claviceps purpurea (Fr.) Tul. or through industrial fermentation processes with other Claviceps. The key factor for biosynthesis is differentiation of a particular mycelial anatomy. Previous study of these fungi from two disparate English grass genera, Spartina and Phragmites, has shown that only mycelia expressing a plectenchymatic sclerotium-like anatomy in specific axenic culture conditions elaborated ergot alkaloids, and then only as far as lysergic acid. The present report describes sequential cycles of axenic and parasitic cultivation for wild isolates from Dactylis and Alopecurus with intervention of a single ascospore step. This confirms the homozygous character of C. purpurea and defines several potential experimental axenic and parasitic conditions within the species for comparing genomic aspects of partial or full biosynthesis of cyclic tri-peptide alkaloids. Whereas Alopecurus ergot isolates readily parasitized rye, use of Dactylis isolates as inoculum for rye ovaries failed to cause the usual sphacelial fructification but supported growth of exceptionally thin sclerotia, sometimes two in a floret, with low alkaloid content attributed to reduced medullary component. However, after two cycles of axenic and rye-parasitic cultivation, and consistent re-selection of the plectenchymatic character in axenic mycelia, typical growth of ergot sclerotia occurred on rye. Caution thus seems necessary in tests for putative host specificity in any taxonomic realignments within the classical concept of C. purpurea. A Dactylis ergot isolate was also uniquely shown to parasitise the plumule of germinating rye seeds confirming the susceptibility of apical tissues. A key biosynthetic feature of a mycelial glyceride oil, rich in ricinoleic acid, as a prelude to axenic and parasitic formation of ergot alkaloids by C. purpurea is emphasised.

The important ergot alkaloid intermediate chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the combined action of EasC and EasE from Aspergillus japonicus.

BackgroundErgot 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.ResultsThe 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.ConclusionsA 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 materialThe online version of this article (doi:10.1186/s12934-014-0095-2) contains supplementary material, which is available to authorized users.

Physiological controls and regulation of ergot alkaloid formation

Innovation and technical development of ergot alkaloids (EA) has moved closer to scientific research. Circumstantial evidence presently links the initiation of EA metabolism to changes in a range of parameters--morphology, concentrations of enzymes and their substrates, nutrients and external stress. The biosynthesis of EA begins at the level of the genetic information apparatus and continues at the level of physiological expression. EA and their formation play a role in the physiology of the production organism. Insufficient insight into Claviceps physiology hampers the deployment of computers in the control and regulation of the EA process. Knowledge of physiological controls and genetic manipulation are the principal tools of modern EA production. In principle it is now possible to improve EA yields by a concerted breeding of the ergot fungus by sexual and parasexual genetic engineering.

Regulation of secondary metabolism in fungi

Abstract

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (Fr.) Tul.

AbstractL‐Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine‐accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high‐yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mm stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5‐Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (FR.) Tul.

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

End-Product Regulation of Ergot Alkaloid Formation in Intact Cells and Protoplasts of Claviceps Species, Strain SD 58

End-Product Regulation of Ergot Alkaloid Formation in Intact Cells and Protoplasts of Claviceps Species, Strain SD 58

Clavicipitic acid: its structure, biosynthesis, and role in ergot alkaloid formation

Clavicipitic acid: its structure, biosynthesis, and role in ergot alkaloid formation

Lysergylpeptides in the course of peptide ergot alkaloid formation.

Radioactive d-lysergyl-Val-Leu-OMe, d-lysergyl-Val-Val-OMe and d-lysergyl-Val-Val-Pro-OMe were synthetized according to the dicyclohexylcarbodiimide/1-hydroxybenzotriazole procedure. These compounds are not used by intact mycelium of Claviceps as immediate precursors for cyclolalkaloid biosynthesis.

Thermodynamic considerations in the formation of ergot alkaloids

Thermodynamic considerations in the formation of ergot alkaloids