Vladimír Křen

Physiological activity of immobilized cells of Claviceps fusiformis during long-term semicontinuous cultivation

SummaryThe 550-day semicontinuous cultivation of Claviceps fusiformis immobilized in calcium alginate is documented. The vegetative mycelium from seed or from early-production submerged culture is the best choice for immobilization. No extracellular glucans are produced by immobilized cells. Immobilized spores give low yields of clavine alkaloids. Alginate concentrations in a range of 2%–4% do not influence yield and spectrum of alkaloids. The cytoplasm of the immobilized cells becomes condensed (after 3 days), polysaccharides disappear, and centres of lipid synthesis are formed in the cytoplasm. After 60 days the cells harbour a great number of lipid particles, mitochondria are diminishing and their cristae partly disappear, indicating a decreased respiration capacity. After 350–500 days the volume of most cells is increased many times and the cells are filled with large oval bodies of electrondense material. Chloramphenicol protects immobilized cultures against bacterial contamination.

Glycosylation of ergot alkaloids by free and immobilized cells of Claviceps purpurea

SummaryA new strain, Claviceps purpurea 88-EP-47, with high invertase activity was selected. Free and Calginate immobilized cultures of this strain were used for fructosylation of ergot alkaloids. By bioconversion from their aglycones, elymoclavine-O-β-d-fructofuranosyl(2→1)-O-β-d-fructofuranoside, and elymoclavine-O-β-d-fructoside, the respective fructosides of chanoclavine, lysergol and dihydro-lysergol monofructosides were obtained. These substances are formed by β-d-fructofuranosidase present in Claviceps cells. The bioconversion activity of the enzyme system (fructose transfer) is strongly dependent on pH, substrate (sucrose) concentration and the developmental profile of invertase activity. The pH optimum for elymoclavine fructosylation is 6.5, for chanoclavine 5.7, and the optimal sucrose concentration is 75 g/l. Fifteen-day-old production cultures had the best glycosylation activities. Fructosylation of alkaloids can be stimulated in production cultures of C. purpurea or C. fusiformis forming elymoclavine or chanoclavine by a pH shift to 6.5 at the end of the production phase. Glycosylating Claviceps strains producing elymoclavine eliminate the free alkaloid into glycosides. The feedback inhibition of alkaloid synthesis by elymoclavine is then strongly reduced, helping to further improve elymoclavine yields. Elymoclavine can be liberated simply by invertase activity of bakers yeast.

Identification of sterols in submerged cultures of different Claviceps species

Summary The occurence of sterols was studied by gas chromatography - mass spectrometry in the lipid fraction of saprophytic cultures of Claviceps purpures, C. paspali, C. fusiformis and C. sp. SD-58. A total of 15 sterols were found out of which 11 have not yet been described in the genus Claviceps . Comparison of biogenetic pathways of sterols was used to assess the relatedness of the strains under study.

Effect of clomiphene on fatty acids, sterols and membrane fluidity in clavine producing claviceps purpurea strains

Clomiphene depressed the growth and enhanced clavine production of Claviceps purpurea strains 129,35 and 59. Mycelial content of 18:2 and 16:0 fatty acids decreased, whereas that of 18:1 and 18:0 acids increased. In the mutant strain 59 clomiphene, triadimefon and ergosterol stimulated the impaired function of chanoclavine cyclase. Their effect was counteracted by plant oil. Clomiphene decreased the content of total lipids (44%), triglycerides (32%), sterols (22%) and sterol/phospholipid molar ratio. The PC/PE ratio was 9X increased. Clomiphene and triadimefon enhanced membrane fluidity of protoplasts, ergosterol and oil reverted their effect.

Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

SummaryKinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both “elymoclavine-inhibitionless” and “clavine alkaloid-decompositionless” fermentations if a combination of fermentation and separation units in a closed loop was used.

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 triadimefon on lipids, sterols, and membrane fluidity in submerged cultures of claviceps purpurea

Abstract Triadimefon (TDF) (5 mg · liter−1) stimulated the functions of chanoclavine cyclase in submerged cultures of high producing mutant Claviceps purpurea 59 with partially blocked oxidation and cyclization of chanoclavine. The culture growth decreased and specific clavine production was raised. The effect of TDF on growth and production was reversed by plant oil (7.2 g · liter−1), but not by ergosterol (10 mg · liter−1). The oil stimulated growth and lowered both specific alkaloid production and chanoclavin cyclase function. Ergosterol showed the opposite effects. TDF reduced the total mycelial lipids to 66%, sterols to 40%, and phospholipids to 72%. In the cultures supplemented with ergosterol, the sterol content increased 16-fold and phospholipids were lowered to 27%; however, the total lipid content remained unchanged. Oil increased all lipid components. The sterol content was indirectly proportional to the PC PE ratio in all cultures. The membrane fluidity of protoplasts from TDF-treated cultures was higher, in ergosterol-treated cultures lower, and unchanged in the presence of oil. The content of methylated sterols in the TDF-treated cultures was only 2% higher than in controls. The demethylation of sterols in Claviceps occurs first in position C-14, then in C-4. The chanoclavine cyclase function probably does not depend on membrane fluidity, but a correlation between higher activity and low phospholipid content was found.

Saprophytic production of clavine alkaloids and activity of hydroxymethylglutaryl-CoA reductase.

In submergedClaviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.

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.

Regulation of Lipid and Ergot Alkaloid Biosynthesis in Claviceps purpurea by Chlorophenoxy Acids

Summary Lipids influence ergot alkaloid biosynthesis in Claviceps purpurea mainly as structural elements of membranes. Chlorophenoxy acids (CPA) were applied to C. purpurea cultures to modify cellular lipid composition. Mutant strain C. purpurea 59 accumulating tricyclic clavines and two parental strains 35 and 129 were used. All the CPA - clofibrate, 2-(4-chlorophenoxy)-acetate and 2-(4-chlorophenoxy)-propionate (CPP) lowered lipid content. Fatty acid (FA) unsaturation index increased indicating a stimulation of FA-desaturation and also FA-elongation. Sterol biosynthesis was impaired mainly in the C(4)- and C(14)-demethylation resulting in a decrease of “membrane functional” (quasiplanar) sterols. Relative content of phosphatidylethanolamine decreased 1 Of old. Changes in lipids correlated with the decrease of microviscosity of cell membranes (measured as a steady state fluorescence anisotropy of the protoplast membranes with DPH and TMA-DPH as probes). CPP in subtoxic doses stimulated alkaloid production and changed alkaloid composition in the C. purpurea 59 strain impaired in chanoclavine cyclase function.