Biosynthesis of ergotamine by Claviceps purpurea (Fr.) Tul

1. Partially purified ceramide trihexoside alpha-galactosidase from human liver was studied by using ceramide trihexoside specifically tritiated in the terminal galactose. 2. The hydrolysis of ceramide trihexoside was absolutely dependent on a mixture of sodium taurocholate and Triton X-100 and was markedly inhibited by human serum albumin and by NaCl. 3. The Lineweaver-Burk plot for ceramide trihexoside hydrolysis was upward curving. Ceramide lactoside inhibited hydrolysis of all concentrations of ceramide trihexoside. Ceramide digalactoside stimulated hydrolysis of low concentrations of ceramide trihexoside, but inhibited hydrolysis of high concentrations of the lipid. 4. alpha-Galactosidase activity assayed with the synthetic substrate 4-methylumbelliferyl alpha-d-galactopyranoside fractionated together with activity assayed with the natural substrate ceramide trihexoside. Both activities had identical heat-inactivation kinetics. 5. Characteristics of the hydrolysis of the synthetic substrate differed considerably from those of the natural substrate, including pH optimum, shape of the Lineweaver-Burk plot, and differential effects of inhibitors and activators. Mutual inhibition of hydrolysis between the synthetic and natural substrates was predominantly non-competitive. 6. These results are discussed in the light of special problems involved in the hydrolysis of lipids in an aqueous milieu.     

Studies on Claviceps purpurea (Fr.) Tul. parasitic on Phragmites communis Trin

Isolates from C. purpurea sclerotia occurring naturally on Phragmites communis usually sporulated vigorously on the culture medium employed, and their failure to produce alkaloid in vitro was associated with a thin white growth form. Such isolates also failed to produce sclerotia on the host plants tested. A variant having a plectenchymatic morphology in vitro and producing a thick pigmented non-sporulating growth form yielded alkaloid (up to approximately 300μg/ml mainly δ8–9 and δ9–10 lysergic acids and chanoclavine) in surface or submerged culture and developed typical ergot sclerotia (containing 0·2-0·4% alkaloid, mainly ergotoxine and ergotamine) in vivo. Improved alkaloid yield in vitro was obtained from a strain reselected after passing through a parasitic phase. Aetiological aspects of the P. communis ergot are discussed.     

Isolation of the Ergot Strain Claviceps purpurea(Fr.) Tul. VKM-F-3662D Producing the Lactamic Alkaloid Ergocornam

A new ergot strain, VKM-F-3662D, producing lactamic alkaloid ergocornam with concomitant alkaloids valinamide and ergometrine, was isolated during selective work with sclerotium MS-462, which was obtained from ergocryptine ergot strain VKM-F-2642D. The structure of these alkaloids was determined by ¹H and ¹³C NMR.     

Improved medium for saprophytic production of ergot alkaloids by Claviceps purpurea (Fr.) Tul

Studies were undertaken on the production of ergot alkaloids in saprophytic culture employing two strains of C. purpurea. In an attempt to improve the yield of the alkaloids and to develop a cheaper medium, the commonly used carbohydrate source, mannitol, was replaced with different types of starches and starchy materials as these are comparatively much cheaper than mannitol. Results indicated that, in stationary cultures, certain starches enhanced the yield greatly, while in shaker cultures starches could replace mannitol for equivalent yields.     

Emergence and phytopathological properties of a new strain of Claviceps purpurea (Fr.) Tul. on rye

The emergence of a new stable strain of Claviceps purpurea is described. The strain formed the sphacelial stage very weakly on rye, and was unlikely therefore to be able to initiate an epidemic under field conditions, but the sclerotia contained chanoclavine as the major alkaloid in contrast to the ergotamine component of the parent strain. Isolates of C. purpurea derived from ryegrass ergots were also shown to parasitize rye.     

Changes of the cytoplasmic ultrastructure during development of sclerotia in Claviceps purpurea (Fr.) Tul.

The development of sclerotia of Claviceps purpurea was investigated by light and electron microscopy. During the first days after infection sterigma and conidiospores are formed. The spores show a moderately developed vacuolar system, they are thick walled and contain about 20% lipid (related to the cell volume) embedded in glycogen. The sterigma are cylindrical unicellular hyphae with electron dense cytoplasm and isolated strongly contrasted lipid droplets. In maturing sclerotia the hyphae become septated with increasingly thick cell walls and a large lipid content. The lipid forms small droplets in young cells, while in the mature sclerotium it occurs in the form of very large drops, occupying the major part of the cell. Simultaneously the composition of the lipid is changed. The mature cells have several nuclei. They are partially connected by osmiophilic substances, forming a network of intercellular spaces.Abbreviations HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - DMSO Dimethylsulfoxide     

Heterokaryosis and Alkaloid Production in Claviceps purpurea

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, is a heterokaryon; after several generations on agar media, it segregates the single components. These components (by us labeled V, C, and W) are stable and produce almost no alkaloids under described conditions of submerged culture. The mycelium of strain 275 FI consists of hyphae with multinucleate cells and does not produce conidia. Strains V, C, and W form numerous anastomoses when grown together on agar. By combining strains V and C, a heterokaryon similar to 275 FI in appearance has been obtained. This new strain produces amounts of alkaloids much larger than those produced by V and C separately or in associated submerged culture. We conclude, therefore, that in strain 275 FI the heterokaryotic condition is favorable to the production of alkaloids. Several conidiaproducing cultures of C. purpurea of various origins, as well as sclerotia of the same species, have been examined. The results demonstrated that the heterokaryotic condition is rare in the cultures, but it is frequent in the mycelium from sclerotia. Since it is known that the production of alkaloids is typical of the sclerotial phase in C. purpurea, it is suggested that this capacity is related to the heterokaryosis of the producing strains.     

The ultrastructure of spores ofClaviceps purpurea (Fr.) Tul.

The ultrastructure of saprophytic and parasitic spores of the AscomyceteClaviceps purpurea (Fr.) Tul. was studied. Considerable differences were found to exist between the saprophytic and parasitic spores as to morphology and fine structure. The reason for the different ultrastructural morphology is probably connected with the intensity of cell metabolism. Whereas the parasitic spores obtained from the honeydew possess the character of a resting cell with a thick electron-dense cytoplasm, abundant lipid bodies, few mitochondria, an underdeveloped and hence little active endoplasmic reticulum and with a homogenous thick cell wall, the saprophytic spores appear as cells with higher metabolic rate, containing more numerous mitochondria, a thinner cytoplasm, a highly developed endoplasmic reticulum, fewer lipid bodies and abundant large vacuoles as well as frequently a new wall layer.     

The cellular role of nitrogen in the biosynthesis of alkaloids by submerged culture of Claviceps purpurea (Fr.) Tul.

Asparagine was a superior nitrogen source for clavine-alkaloid production in Claviceps purpurea. Its transport into the cell excedded the cell's biosynthetic need for this amino acid. Asparagine entered the cell without degradation. This disturbed the relative pool sizes of various amino acids resulting in a change in the genetically determined ratio at which amino acids were utilized for protein synthesis. Overproduction of alkaloids (4500 mug.ml-1) may be associated with increased availability of tryptophan because of the enhanced assimilation of asparagine-derived ammonia via glutamine synthetase (EC 6.3.1.2). However, ammonium salts in the fermentation broth led to a depression of the alkaloid yield. Partial replacement of the ammonium salt by aspartic acid elevated alkaloid production.     

Morphological differentiation of cells in submerged cultures of Claviceps purpurea (Fr.) Tul., producing alkaloids]

Differentiation of the cells of the submerged culture of Claviceps purpurea (Fr.) Tul. was studied by electron microscopy. Two types of oviform cell were found: (1) the conidia which had one nucleus and vacuolized cytoplasm and were not involved in the production of alkaloids; (2) the chlamydospores with two nuclei, homogeneous cytoplasm, and high content in lipids. The chlamydospores, like the cells of sclerotia, were found to produce alkoloids.     

Production of alkaloids and differentiation in a submerged culture ofClaviceps purpurea (Fr.) Tul

The submerged culture of the Claviceps purpurea strain studied was polymorphous. In the process of alkaloid synthesis, cytodifferentiation preceded biochemical differentiation. The onset of the alkaloid phase was characterized by: predominance of chlamydospores in the culture, the presence of vegetative cells with reduced or arrested proliferation, maximum acetylCoA carboxylase activity, the maximum amount of total fatty acids, an over-average cell pool tryptophan level and minimum tryptophan synthetase activity. Intracellular ricinoleic acid was an indicator of differentiation of the culture towards alkaloid formation and also of alkaloid synthesis. The cytodifferentiation period in the initial phases of fermentation, when the cell has several alternative possibilities of development, is regarded as the most sensitive sector of the regulatory mechanisms of alkaloid formation.     

Isolation of the ergot (Claviceps purpurea (Fr.) Tul., strain VKM-F-366D), producing the lactamic alkaloid ergocornam

A new ergot strain VKM-F-3662D producing lactamic alkaloid ergocornam with concomitant alkaloids valinamide and ergometrine was isolated during selective works with sclerotium MS-462, which was obtained from ergocryptine ergot strain VKM-F-2642D. The structure of these alkaloids was determined by 1H and 13C NMR.