Strain Of Claviceps Purpurea Research Articles

Developmental studies of Claviceps paspali seed cultures for the submerged production of lysergic acid derivatives

Metabolic pattern of mycelial Claviceps paspali seed cultures during the submerged cultivation was established. By comparing it with conidial and mycelial Claviceps purpurea strains it was found that the biosynthesis of RNA, DNA, and proteins followed a similar course in all Claviceps strains, so the fall of RNA content in mycelium may be considered a general biochemical indicator for optimally developed inoculum. But, two different patterns of carbohydrate and lipid metabolism were observed one for conidial and one for mycelial strains.

Role of the δ 8 double bond of agroclavine in lysergic acid amide biosynthesis byClaviceps purpurea

Agroclavine, given to actively-growing sclerotial tissue of a strain of Claviceps purpurea which can not normally elaborate ergot alkaloids, was transformed by this tissue into lysergic acid amide with overall efficiency of approximately 40%. By contrast, festuclavine (8,9-dihydro-agroclavine) was not transformed, indicating specificity in the mechanism of lysergyl biosynthesis.

Phosphate regulation of phosphatases in submerged cultures of Claviceps purpurea 129 producing clavine alkaloids

Claviceps purpurea strain 129 was cultivated under submerged conditions in a sucrose-citrate medium containing high (36.8 mM) or low (1.84 mM) KH2PO4 concentrations. The permeabilized cells and culture supernatants contained alkaline and acid phosphatases. In the medium containing a high phosphate concentration, the synthesis of extracellular phosphatases was repressed, but that of cellular phosphatases was not. Extracellular phosphatases, especially alkaline phosphatases, were derepressed by transferring the mycelium into a phosphate-free medium. This derepression was inhibited by cycloheximide. In the presence of cycloheximide, the activities of the cellular phosphatases decreased markedly, indicating turnover of these enzymes. The cellular acid phosphatase was inhibited by phosphate (0.025 M–0.1 M) and NaF (0.01 M) while the cellular alkaline phosphatase was only inhibited by phosphate. Both cellular and extracellular alkaline phosphatases were more sensitive to repression by phosphate than the acid phosphatases. The alkaloid synthesizing enzymes were: a) present in mycelia grown in high levels of phosphate and b) activated by decreasing the intracellular phosphate level.

Unusual Ergot Alkaloid Biosynthesis in Sclerotia of a Claviceps purpurea Mutant

A strain of Claviceps purpurea, which has consistently failed to elaborate ergot alkaloids when growing as a parasite, has been shown to perform only the first step of the ergoline biosynthetic pathway catalysed by dimethylallyltryptophan (DMAT) synthetase. The next step, involving N-methylation of DMAT, did not operate. [14C]Agroclavine and lysergic acid, normally intermediates in alkaloid biosynthesis, were accepted by parasitic sclerotial tissue as substrates and were metabolized to lysergic acid amide (LAA). This amide therefore constituted a previously unreported end product for C. purpurea. It is concluded that the mutant has a metabolic block in the pathway following DMAT and, while enzymes for several subsequent steps are present, the fungus seems unable to form the usual cyclic tripeptide ergot alkaloids. The steps involved in metabolizing agroclavine to LAA were insensitive to 1 m-phosphate, while this concentration of phosphate completely inhibited DMAT synthetase. This double mutant therefore has unique potential for exploring control mechanisms in ergot alkaloid biosynthesis.

Extrachromosomal genetics of Claviceps purpurea

In order to establish a system for gene cloning in the ergot fungus Claviceps purpurea mitochondrial DNA was purified from two wild strains and four descendants of production strains. A characterization of the mt DNA with respect to buoyant density, molecular weight (45 kb corresponding to acontour lenght of 14 μm) showed no differences. However, considerable differences were detected in the restriction pattern, with the exception of two commercial strains of possibly common origin. From this it follows that restriction patterns of mt DNA may be used as finger prints to identify related strains.In one of the wild strains plasmids associated with the mitochondria were found. A characterization of two species p11 and p12 having molecular weights of 6.6 and 5.3 kb revealed that these genetic units are linear having respective contour lengths of 2.1 and 1.7 μm. According to Southern blot analyses both plasmids show homology to each other, but not to the mitochondrial "chromosome". Accordingly, p11 and p12, despite localization in the mitochondria, are evidently not an integral part of the mt "chromosome". The possibility to use the mt DNA and mitochondrial plasmids to establish a vector system for molecular cloning in imperfect strains of Claviceps purpurea has been pointed out.

A New Commercial Strain of Ergot Adapted from a Wild Grass

A strain of Claviceps purpurea (Fr.) Tul. isolated from Arthraxon lancifolius Hochst. was successfully adapted on rye. Experimental cultivation on rye showed 370 kg/ha yield of sclerotia containing 0.5% total alkaloid which consisted of 33% ergometrine, 17.6% ergotamine, 18.7% ergocornine and 22.7% ergokryptine. The results also reveal that alkaloid profile of the ergot strain depends largely on its host. Significantly high yield of the sclerotia and the valuable alkaloid profile of the new strain indicate its commercial potential.

Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids‐producing strain of Claviceps purpurea (FR.) TUL

AbstractA strain of Claviceps purpurea, designated Pepty 695/S produces ergotoxine alkaloids under particular conditions of fermentation. The onset of alkaloid synthesis occurs around the second day of cultivation. Alkaloid formation is connected with morphological and ultrastructural changes. In the first 3‐5 days of cultivation short thickened, septated hyphae, organized in plectenchymatic pellets as well as large single cells are formed. The hyphae are ultrastructurally characterized by increasing number of lipid droplets, deposits of glycogen and by extended ER membrances, which apparently may form numerous vesicles. The correlations between lipid and alkaloid synthesis are discussed.

Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea (Fr.) Tul.

A strain of Claviceps purpurea, designated Pepty 695/S produces ergotoxine alkaloids under particular conditions of fermentation. The onset of alkaloid synthesis occurs around the second day of cultivation. Alkaloid formation is connected with morphological and ultrastructural changes. In the first 3-5 days of cultivation short thickened, septated hyphae, organized in plectenchymatic pellets as well as large single cells are formed. The hyphae are ultrastructurally characterized by increasing number of lipid droplets, deposits of glycogen and by extended ER membranes, which apparently may form numerous vesicles. The correlations between lipid and alkaloid synthesis are discussed.

Glucose catabolism during alkaloid production in a strain of Claviceps purpurea.

Badiorespirometric experiments with glucose labelled in positions 1; 2; 3,4 and 6 were carried out inClaviceps purpurea strain pepty 695. The pattern of14CO2 evolution from 5 – 50 mM glucose indicated the operation of the pentose phosphate cycle during alkaloid production.

Mutational Biosynthesis in a Strain of Claviceps purpurea1

An alkaloid-blocked mutant of Claviceps purpurea was isolated from an ergotoxine producing strain. It accumulates chanoclavine I and the corresponding aldehyde. The formation of agroclavine is blocked in this particular strain. By feeding with appropriate intermediates (tetracyclic ergolines) the mutant produces the normal alkaloidal spectrum of the parent strain. Surprisingly, after supplementation with agroclavine, ergosine is synthesized.

Induction of sclerotialike mycelium in axenic cultures of Claviceps purpurea producing clavine alkaloids

Aggregates of sclerotialike (SL) cells, present normally among filamentous sphacelial hyphae of submerged cultures of Claviceps purpurea strain 129 synthesizing clavine alkaloids, may represent an adaptation to unfavorable environmental conditions. Their formation can be induced by dyes (acriflavine, toluidine blue, fluorescein, basic fuchsin, ethidium bromide, and methylene blue). A treatment of the original strain with ethyl methanesulphonate yielded five mutants with stable SL phenotypes. Electron microscopy was used to illustrate the morphogenetic processes in mutant strains and the fragmentation of SL clusters to arthrospores.

Ultrastructure of submerged non-alkaloid producing strains of Claviceps purpurea (Fr.) Tul.

Summary Two non-alkaloid producing strains of Claviceps purpurea were investigated after cultivation in two different media.The ultrastructure of the hyphae depends on the composition, of the culture broth.The strain Pur 221 forms in, the “production” medium NL 720 thin-walled hyphae with several nuclei numerous mitochondria and ER membranes.In older hyphae most of the cell volume is occupied by polysaccharides. The strain Pur 218, grown in a rich medium, shows relatively thin-walled hyphae with large nuclei, short ER membranes and only a small number of lipid droplets.In a “production” medium hyphae of the same age contain 50% lipid related to the cell volume.It can be concluded from the electron micrographs that the composition of the lipids is changed during the cultivation.Morphometric analysis of the most important cell organelles allows a more precise description of the development of the fungus during cultivation and under different environmental conditions.

Influence of Krebs Cycle Acids on Clavine Alkaloid Production and Changes in Activity of Krebs and Glyoxylate Cycle Enzymes During Fermentation

The shake-flask cultures of a Claviceps purpurea strain with high clavine-alkaloid production were investigated. At the early fermentation the Krebs cycle played a dominant role as an amphibolic pathway. The intensive alkaloid synthesis was preceded by a decrease of the Krebs cycle activity and significant rise of the glyoxylate bypass. Pyruvate entered the cell predominatly via glyoxylate bypass. 2-ketoglutarate, isocitrate, citrate, fumarate, malate, and succinate markedly stimulated the alkaloid synthesis. Catabolite repression is assumed to take inhibitory part in regulation of the alkaloid synthesis. Some Krebs cycle acids might participate in overcoming of influence of the catabolite repression. In Schüttelkultur wurde die Clavin-Synthese eines Claviceps-purpurea -Stammes mit intensiver Alkaloidsynthese untersucht. Zu Beginn der Fermentation hat der Krebszyklus erstrangige Bedeutung. Intensiver Alkaloidsynthese ging ein Rückgang der Aktivität des Krebszyklusses und eine Erhöhung der des Glyoxalatzyklusses voraus. Pyruvat tritt in die Zelle hauptsächlich über den Glyoxylatzyklus ein. 2-Ketoglutarat, Isozitrat, Zitrat, Fumarat, Malat und Sukzinat stimulieren die Alkaloidbildung. Die katabolische Repression spielt eine hemmende Rolle bei der Steuerung der Alkaloidsynthese. Einige Säuren des Krebszyklusses beteiligen sich an der Hemmung der katabolischen Repression bei der Alkaloidbildung.

Biosynthesis of Ergot Alkaloids by Protoplasts of Various Claviceps purpurea Strains1

Protoplasts of various peptide type alkaloids producing Claviceps purpurea strains were prepared. In a sucrose ammonium citrate medium suspended protoplasts were able to synthesize de novo ergosine, ergotomine and ergotoxine using 14C-labelled amino acids as precursors. The incorporation rate of the amino acids was remarkably increased if the incubation mixture was supplemented with d-lysergic acid.

Content of the alkaloid sum, ergocryptine, and fatty oil in ergots of an ergocryptinic strain of Claviceps purpurea

Content of the alkaloid sum, ergocryptine, and fatty oil in ergots of an ergocryptinic strain of Claviceps purpurea

Einfluß von Cycloheximid auf Wachstum und Alkaloidbildung bei Claviceps purpurea, Stamm Pepty 695

Cycloheximide (CHI) inhibits in rather low doses (10–20 μ g/ml) growth of Claviceps purpurea strain Pepty 695 and subsequently alkaloid formation in submerged cultures irrespectively of the time of addition. The ability of growth and alkaloid formation is retained after removal of the inhibitor. Protein synthesis is strongly inhibited in replacement cultures but the formation of the peptide portion of ergotoxine is not suppressed. On the contrary in CHI-supplemented cultures a higher incorporation rate of [U- 14 C]-valine into alkaloids was observed in short term experiments. The synthesis of the peptide portion of cyclol alkaloids is apparently a non-ribosomal process. The “cyclol synthetase” seems to be not more than 48 h stable in vivo.

Über die chemische Zusammensetzung der Zellwand von Claviceps-Arten

Summary Hyphal walls were isolated from Claviceps purpurea strains (alkaloid producing; alkaloid non-producing) and Claviceps fusiformis. The fungi were cultivated under submerged conditions in different culture media. The main neutral sugars were glucose, mannose and galactose. The principal sugar accounting for more than half of this particular fraction was glucose. The amino sugar fraction predominantly glucosamine varied in different strains, e.g. in the ergotoxine producer Pepty 695 between 20–37%. The content of the uronic acid fraction amounted to about 5–8% of the wall. Lipids comprise a relative high amount of wall material depending on the strain. The main fatty acids are lauric acid, myristic acid and palmitic acid. Claviceps cell wall material was found to have about 5–10% protein. Protein hydrolysis gave 15 amino acids, predominantly threonine, serine, aspartic and glutamic acid.

Unterschiede im Prolinstoffwechsel eines Peptidalkaloide‐produzierenden und eines nichtproduzierenden Stammes von Claviceps purpurea

AbstractTwo strains (pepty 695 and pur 221) of Claviceps purpurea have been used to study the dependence of proline turnover on incubation time (1, 3, 6, and 12 hours) under different culture conditions (synthetic saccharose‐citrate medium NL 720 and complex wort medium M 107) by means of tracer technique.In the saprophytically ergotoxine producing strain pepty 695 the proline is utilized in the protein and alkaloid biosyntheses. On the other hand, under non‐producing conditions (M 10) the radioactivity of proline‐14C is incorporated into many fractions and finally into CO2. In submerged culture (NL 720) the Claviceps strain pur 221 which does not produce ergolines uses proline only to a small extent in the protein biosynthesis, while most of this amino acid is not metabolized.The extent of uptake of proline is not correlated with the alkaloid synthesis.For comparison the turnover of l‐glutamate‐14C and d, l‐ornithine‐1‐14C by strain pepty 695 cultivated in the NL 720 medium has been studied.

Strain and milieu specific amino acid patterns of Claviceps purpurea during saprophytic cultivation

Strain and milieu specific amino acid patterns of Claviceps purpurea during saprophytic cultivation

Genetics of the ergot fungus Claviceps purpurea

The morphological and physiological evaluation of single ascorspore lines (F1) obtained from 4 production strains of Claviceps purpurea and the subsequent production and evaluation of an F2 from two of these strains has shown: 1. The breeding system responsible for the sexual cycle of C. purpurea is characterized by monoecism and self-compatibility. 2. Heterokaryosis is neither required for the completion of the life cycle nor for alkaloid synthesis. 3. The huge variability in both mycelial morphology and alkaloid spectra makes it evident that all production strains are highly heterogeneous. 4. A correlation between morphology and an alkaloid pattern was not found. 5. Alkaloid production is, however, correlated with distinct genotypes. 6. In some cases an increase in the total alkaloid content and in the formation of specific alkaloids was also found, showing that the genetic control of these metabolites may be accessible. Taken together these results open up the way to concerted breeding with Claviceps purpurea with the object of improving alkaloid production.