ofClaviceps Purpurea Research Articles

Effect of cultivation temperature, clomiphene, and nystatin on the oxidation and cyclization of chanoclavine in submerged cultures of the mutant strainclaviceps purpurea 59

Submerged cultures ofClaviceps purpurea 59 produce predominantly tricyclic chanoclavine and chanoclavine-I-aldehyde; formation of the tetracyclic agroclavine and elymoclavine is limited to 20%. The production cultures were characterized by the oxidation index oi (100% chanoclavine/% chanoclavine) and cyclization index ci (% agroclavine +% elymoclavine/% chanoclavine-I-aldehyde), expressing two functions of chanoclavine cyclase. Both indices were influenced by cultivation temperature and membrane agents. At 20°–24°C, clomiphene increased oi and ci; at 28°C and more it increased oi only. At 24°C nystatin increased oi and decreased ci. During cultivation of vegetative inocula and production cultures at various temperatures (24°, 28°, and 33°C), the lower cultivation temperature of the inoculum increased ci of the production culture with the higher temperature. The higher cultivation temperature of the inoculum decreased oi and particularly ci of the production culture with the lower temperature. In production cultures cultivated from the inoculum of an identical temperature, oi decreased to 50% with increasing temperature above 24°C, whereas ci decreased continuously. The role of the chanoclavine cyclase conformation as a membrane enzyme is discussed.

Effect of immobilization on the stability ofClaviceps purpurea protoplasts

Protoplasts released with high efficiency from vegetative and productive hyphae ofClaviceps purpurea were immobilized in 2% Ca-alginate. The yield of active immobilized protoplasts depended upon the age of the mycelium from which protoplasts were derived and was found to be 25–43% in comparison with native hyphae. During incubation in a modified production medium immobilized protoplasts were stable for at least 10–12 days. No external growth of regenerated hyphae from spherical beads of alginate gel with entrapped protoplasts was observed for 13–15 days of the batchwise incubation.

Effect of immobilization on the stability ofClaviceps purpurea protoplasts

Effect of immobilization on the stability ofClaviceps purpurea protoplasts

Occurrence of ricinoleic acid in submerged cultures of variousClaviceps sp.

Ricinoleic acid was found in differentClaviceps sp., indicating that it is neither specific chemotaxonomic marker ofClaviceps purpurea, nor a specific indicator of peptide alkaloid synthesis.

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.

A blocked mutant ofClaviceps purpurea accumulating chanoclavine-I-aldehyde

An alkaloid-blocked mutant ofClaviceps purpurea was isolated from a strain which produces ergotoxine alkaloids. The mutant accumulates chanoclavine-I and the corresponding aldehyde. It lacks the ability to form tetracyclic ergolines.

The effects of maintained tryptophan levels on ergot alkaloid production and overall tryptophan economy inClaviceps purpurea

Measurements of alkaloid production, dryweight, and total tryptophan requirement in batch cultures ofClaviceps purpurea maintained at predetermined levels of extracellular tryptophan by repeated measured additions of the aminoacid confirm previous accounts of the overall regulatory effects even in the absence of major fluctuations in the tryptophan level.

Carbohydrate nutrition of claviceps purpurea for alkaloid production related to the osmolality of media

The effects of most frequently used carbohydrates and osmolalities of media on the growth and productivity ofClaviceps purpurea in submerged cultures were investigated. The maximum alkaloid yield was obtained with 20% mannitol as well as with 10% mannitol when 2% NaCl was added to the medium and the osmolality about 1 osmol/kg H2O was established at the end of fermentation. Increased or decreased osmolality of the medium had a negative influence on fungal growth and alkaloid production. Almost the same effect was observed with sucrose as with mannitol, whereas with glucose only maximal growth was achieved.

Production of beta-ergokryptine by a strain of Claviceps purpurea (Fr.) Tul. in submerged culture.

A strain ofClaviceps purpurea, labelled 231 F.I., produced in submerged culture 1200 μ/ml of a mixture of peptidic alkaloids composed for 30% by β-ergokryptine. Cultural media, conditions of culture and development features of a typic fermentation are reported.

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.

On the incorporation of dipeptides in ergotoxin alcaloids

l-Valyl-l-leucine-U-14C,l-valyl-l-proline-U-14C-lactam andl-leucyl-l-proline-U-14C-lactam were administered to submerged cultures of an ergotoxine-mixture-producing strain ofClaviceps purpurea. The added dipeptides were split by the fungus into the corresponding amino acids prior to incorporation. Apparently dipeptides are no immediate precursors for the peptide side-chain of ergotoxinealkaloids.

Experimental cultivation of ergot at Jorhat, Assam and extraction of alkaloids

Climatic conditions of Jorhat, a high percentage of relative humidity and low termperature during January, February are highly favourable for the infection and yield of ergot at Jorhat. The best time of sowing rye is the first fortnight of October and artificial infection during January. Out of 15 strains ofClaviceps purpurea used in this study, the “ergometrine” strain and “ergotamine” strain, received from the University of Johannes, Gutenberg, Mainz, performed most efficiently under the condition at Jorhat, giving high yields of sclerotia and alkajoids. The sclerotia produced by these strains were also larger in size than the average.

Influence of auxins on growth of Claviceps purpurea (Fries) Tulasne in saprophytic cultures.

A 10−3 m β-indolebutyric and 2,4-dichlorophenoxyacetic acid concentration stimulated the fresh weight increase and dry matter production in the mycelium of staticClaviceps purpurea cultures to 170–180% of the control value. Higher concentrations severely inhibited growth. α-Naphthylacetic acid was less active. β-Indoleacetic acid differed markedly from the other auxins, as it stimulated the fresh weight increase, but at the same time inhibited dry matter production. In submerse cultivation, the differences between the test auxins disappeared; none of them stimulated growth and only inhibition of growth, by concentrations of over 10−5–10−4 m, was observed. The used strain did not produce alkaloids in saprophytic cultures, even in the presence of auxins.

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.

Ergotamine production in submerged culture and physiology of Calviceps purpurea.

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, and strains V, C, and W, spontaneously obtained from 275 FI and practically unable to produce alkaloids, were compared. Strain 275 FI differs from the other strains in its capacity to accumulate lipids and sterols, as well as in its capacity to produce alkaloids. Strain 275 FI utilizes large quantities of sucrose and citric acid simultaneously; strain V utilizes large amounts of sucrose but little citric acid; strain C utilizes large quantities of citric acid but only small amounts of sucrose; strain W consumes only small amounts of both substances. We conclude that the production of large quantities of alkaloids, as well as the accumulation of lipids and sterols, is correlated with the simultaneous utilization of large amounts of sucrose and citric acid.

The structure of the glycerides of ergot oils

The oils from sclerotia or from suitable mycelial cultures ofClaviceps purpurea (ergot) contain up to 44% of ricinoleic acid but no free hydroxyl groups. This is due to the presence of, besides normal triglycerides, tetra-acid, penta-acid and hexa-acid triglycerides. These contain respectively one, two and three ricinoleic acids esterified to glycerol, these in turn being acylated at their hydroxy groups with normal long-chain fatty acids. By suitable complementary use of TLC, GLC and lipase hydrolysis techniques, the proportions, compositions and structures of these novel triglyceride classes were determined. Four types of positional specificities in fatty acid combinations could be shown by our procedures. These are discussed and, on the basis of our results, some tentative proposals as to possible biosynthetic mechanisms are advanced.

Hypercapsulated growth of Leuconostoc mesenteroides in the sphacelial stage of Claviceps purpurea.

The occurrence in rye florets of an unusual saprophytic growth ofLeuconostoc mesenteroides in the honeydew of the sphacelial stage ofClaviceps purpurea is described. The bacterial infection prevents the further development of ergot sclerotia by interposing hypercapsulated cells in the floral cavity at the base of the ergotised ovary. The capsular material has been identified as the α 1–6 linked glucosan, dextran, and the bacterial growth form has been reproduced artificially in vivo.Saccharomyces kluyveri has been isolated from a naturally occurringL. mesenteroides growth.

Comparison of alkaloid formation of Claviceps purpurea in parasitic and saprophytic cultures

Three monoconidial strains ofClaviceps purpurea were grown parasitically on rye and saprophytically on a nutrient medium. The qualitative alkaloid spectra were nearly identical in the sclerotia and in the corresponding saprophytic cultures, while the quantitative distribution of some of the alkaloids differed considerably.

Glutamic decarboxyiase of ergot, Claviceps purpurea.

Anderson, John A. (Oregon State University, Corvallis), Vernon H. Cheldelin, and Tsoo E. King. Glutamic decarboxylase of ergot, Claviceps purpurea. J. Bacteriol. 82:354-358. 1961.-l-Glutamic acid is the only naturally occurring amino acid which can be decarboxylated by cell-free extracts of Claviceps purpurea. This decarboxylase was partially purified and the properties of the enzyme studied. The specific activity of the purified preparation was 111 muliters per 10 min per mg of protein. The products formed, stability, inhibition, stimulation of activity with pyridoxal phosphate, and pH activity curve were typical of l-glutamic decarboxylase in Escherichia coli and other microorganisms. The substrate constants at pH 4.6, 5.25, and 5.65 were 0.0169 m, 0.0174 m, and 0.0139 m, respectively. The respective maximal velocities at these pH values were 104, 104, and 90 muliters per 10 min. The pH optimum was 4.8 to 5.2. The enzyme was unstable below pH 4.5 and it was suggested that the fall in activity at the lower end of the pH curve was due to inactivation of the enzyme. The decrease in activity above pH 5.2 did not appear to be due to a change in affinity of enzyme for substrate but to a change of the enzyme-substrate complex into an inactive form.