Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58.

Addition of biotin to culture medium NL-406 significantly increased alkaloid yield during submerged cultivation of Claviceps sp. strain SD-58. Alkaloid yield was further enhanced by incorporating leucine in biotin-supplemented culture medium. Increased alkaloid production was associated with an increase in the lipid content of cells and in the number of chlamydospores. Biotin deficiency caused a reduction in alkaloid yield and a parallel decrease in lipid content and chlamydospore numbers.     

Activities of catabolic pathways and alkaloid biogenesis during submerged cultivation ofClaviceps sp. SD-58

Alkaloid biogenesis inClaviceps sp. SD-58 was found to be associated with low growth and reduced consumption of sucrose and mannitol. The activities of glucose-6-phosphate dehydrogenase and aldolase increased up to 9 d of the fermentation cycle and then declined. A close parallel between the operation of pentose phosphate and glycolytic pathways and tryptophan content was demonstrated. An inverse relation between the operation of citric acid and glyoxylate cycles and the ability of the cells to synthesize alkaloids was noted. The role of carbon metabolic pathways during fermentative production of alkaloids is discussed.     

Induced parasexual processes in Claviceps sp. strain SD58

A homokaryotic, clavine alkaloid-producing strain of ergot, Claviceps sp. strain SD 58, was used in an attempt to demonstrate parasexuality. Genetically marked auxotrophic strains were produced by mutation with N-methyl-N'-nitro-N-nitrosoguanidine. Protoplast fusion of pairs of unlike doubly auxotrophic strains and isolation of stable prototrophic fusion products were carried out. By growth of the fusion products on complete medium, selective pressure for prototrophy was removed and auxotrophic segregants were allowed to form. Analysis of these and recovery of segregants with nonleaky, non-parent-type combinations of auxotrophic characteristics has provided strong evidence that a parasexual cycle can function in Claviceps sp. strain SD 58. Preliminary work suggests that the genetics of ergot might be studied by mitotic analysis and that protoplast fusion and selection procedures might be useful for the enhancement of favorable characteristics in Claviceps strains.     

Induced parasexual processes in Claviceps sp. strain SD58.

A homokaryotic, clavine alkaloid-producing strain of ergot, Claviceps sp. strain SD 58, was used in an attempt to demonstrate parasexuality. Genetically marked auxotrophic strains were produced by mutation with N-methyl-N'-nitro-N-nitrosoguanidine. Protoplast fusion of pairs of unlike doubly auxotrophic strains and isolation of stable prototrophic fusion products were carried out. By growth of the fusion products on complete medium, selective pressure for prototrophy was removed and auxotrophic segregants were allowed to form. Analysis of these and recovery of segregants with nonleaky, non-parent-type combinations of auxotrophic characteristics has provided strong evidence that a parasexual cycle can function in Claviceps sp. strain SD 58. Preliminary work suggests that the genetics of ergot might be studied by mitotic analysis and that protoplast fusion and selection procedures might be useful for the enhancement of favorable characteristics in Claviceps strains.     

Effect of tween series surfactants on alkaloid production by submerged cultures of Claviceps species

Supplementation of Tween surfactants promoted alkaloid production by submerged cultured of Claviceps sp. strain SD-58. Tween 80 (0.5%) exhibited the maximum (2-fold) stimulatory effect when added to the medium at the initial stage of cultivation. The stimulation of alkaloid production by Tween 80 was found to be associated with the increase in cell mass, higher consumption of nutrients and enhanced excretion of alkaloids from the cells. The results are discussed in relation to the physiology of alkaloid production in Claviceps sp. strain SD-58.     

Alkaloid production byClaviceps sp. SD-58; Involvement of phosphatase isozymes

Simultaneous reduction in alkaloid yield and level of phosphatases by high concentrations of phosphate was observed inClaviceps sp. SD-58. Tryptophan-induced culture showed an increase in alkaloid yield and the level of phosphatases. Phosphate caused repression of both acid phosphatase (isoenzyme I) and alkaline phosphatase (isoenzymes III and V).     

Alkaloid production during the cultivation with shaking of Claviceps sp: Effects of asparagine

Among the nitrogen sources tested, asparagine stimulated alkaloid production maximally. Ammonium salts supported alkaloid production poorly. During the cultivation with shaking of Claviceps sp. strain SD-58 in asparagine containing medium, the activity of asparagine increased during the exponential growth (up to 8 days) with the intracellular accumulation of ammonium ions. Among the ammonia-assimilating enzymes we studied, NADP⁺-glutamate dehydrogenase (GDH) had a higher activity in the growth phase (up to 6 days), while in the intensive alkaloid producing phase (after 6 days) the activity of glutamine synthetase was higher. The latter was associated with increases in the intracellular level of tryptophan and alkaloid production.The levels of NADP⁺- and NAD⁺-alanine dehydrogenases and glutamate synthase were negligible.     

Metabolic changes in a conidia-induced Claviceps paspali strain during submerged fermentation

Chemical changes in the mycelium of the conidial Claviceps paspali mutant strain, isolated after gamma irradiation, were followed during the course of submerged fermentation and compared with the mycelial parent strain; both strains are capable of producing simple lysergic acid derivatives. The syntheses of lipids, carbohydrates, phosphates, nucleic acids, proteins, and alkaloids, as well as nutrient uptake, were determined. It was found that conidiation induced by mutagenic treatment was accompanied by a set of changes in the metabolic pattern. In the conidial mutant, the primary and secondary metabolic activities were repressed and the protein to nonprotein compound ratio of the cells was changed in favour of protein compounds.     

A mathematical model of growth and alkaloid production in the submerged culture of Claviceps purpurea

A mathematical model of the batch cultivation of Claviceps purpurea was formulated. The main attention was devoted to the effect of exocellular and intracellular phosphate on the growth of the mycelium and production of clavine alkaloid under experimental conditions without limitation by carbon and nitrogen sources. The method of nonlinear regression was used ot predict the optional technological regime of the phosphate addition in the batch culture at different time intervals of additions.     

Sugar and phosphate metabolism and alkaloid production phases in submerged cultures of two Claviceps strains

Summary In submerged cultures of Claviceps sp. CP II, elymoclavine was synthesized only by the growing mycelium (phase P1), whereas cultures of C. purpurea strain 129 produced agroclavine after vegetative growth had also ceased (phase P2). In strain CP II, the peak of activity of malate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphatases was related to the time of maximum growth rate and alkaloid production. Citrate synthase activity paralleled the course of alkaloid synthesis. Strain 129 exhibited a further activity peak of the same magnitude during phase P2. ATP levels in both cultures corresponded to the pattern of change in enzyme activities. Strain CP II contained roughly twice as much orthophosphate and ATP in its cells as strain 129 and exhibited higher average activity of glucose-6-phosphate dehydrogenase. It follows from these results that alkaloid synthesis requires the processes of primary metabolism, even when it occurs after active growth of the culture has ceased. Cultures producing alkaloids oxidized at C-8 exhibit higher glucose-6-phosphate dehydrogenase activity, probably because of a higher NADPH consumption.     

Dehydrogenase activity and toxinogenesis of a production strain ofCorynebacterium diphtheriae during submerged cultivation

A simple biochemical procedure was obtained for studying metabolism ofCorynebacterium diphtheriae during submerged cultivation based on the modification of the assay of dehydrogenase activity using 2,3,5-triphenyltetrazolium chloride as redox indicator. Results obtained by the estimation of the dehydrogenase activity using TTC are in a good accordance with oxygen consumption assayed manometrically. By following dehydrogenase activity in submerged cultivations of a production strain ofCorynebacterium diphtheriae PW8-Weissensee we found that a massive toxin production is connected with the decrease of the activity of cells. This fall of activity occurs yet during the exponential phase of growth. Especially a sudden fall of succindehydrogenase activity exactly indicates the beginning of a considerable toxin accumulation in the medium. The presence of inhibitory concentrations of iron ions in the medium not only increases the level of dehydrogenase activity but changes its whole kinetics. A retarded and irregular fall of the activity occurs instead of a sharp one typical for good toxin production.     

The effect of aeration and agitation on Claviceps purpurea dimorphism and alkaloid synthesis during submerged fermentation

Summary Varying the air flow rate (vvm) in a fermentor under constant drive speed, Claviceps purpurea dimorphism as well as alkaloid biosynthesis were greatly influenced. At a high flow rate (2.5 vvm) sclerotial growth was favoured in seed and in production media, while at a low air flow rate (1.0 vvm) sphacelial growth dominated. When using high flow rates the oxygen uptake rate was small, but at low flow rates it increased markedly. In both cases the alkaloid production was lower than at the intermediate value of 1.5 vvm of air flow rate, which proved to be optimal. This could be explained by the difference in the air/water interface and two-phase oxygen uptake. At a high air/water interface direct oxygen uptake from the gaseous phase prevails, while at a low air/water interface uptake is due to the oxygen liquid-phase only. Thus for optimal fungal development and alkaloid production a compromise between uptake from the liquid and the gaseous phase has to be established by a defined ratio between aeration and agitation.     

Optimization of conditions for storage and cultivation of the fungus Claviceps sp.--a producer of the ergot alkaloid agroclavine

Conditions of agroclavine biosynthesis by the mutant Claviceps sp. strain s 106 were studied. The content of agroclavine was maximum (1.5-2 g/l) on days 15-16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract. Agroclavine was the major component of the alkaloid fraction (90-95%). Storage of the culture at -70 degrees C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.     

添加生物素和浅蓝菌素对裂殖壶菌发酵产DHA的影响

为提高发酵产量,根据裂殖壶菌生物合成二十二碳六烯酸(DHA)的途径,考察添加代谢途径关键酶的辅酶及酶的抑制剂对发酵裂殖壶菌的影响.结果表明:添加生物素可促进油脂积累,添加浅蓝菌素有利于DHA及不饱和脂肪酸含量的提高.添加生物素0.3 mg/L时,DHA占细胞干质量分数达11.26％,相对于对照提高了13％;当添加浅蓝菌素0.1mg/L时,DHA占细胞干质量分数可达12.2％.     

Purification and properties of dimethylallylpyrophosphate: Tryptophan dimethylallyl transferase,the first enzyme of ergot alkaloid biosynthesis in Claviceps. sp. SD 58

Dimethylallylpyrophosphate:l-tryptophan dimethylallyltransferase (DMAT synthetase), the first pathway-specific enzyme of ergot alkaloid biosynthesis, has been isolated from mycelia of Claviceps sp., strain SD 58, and purified to apparent homogeneity. The enzyme reaction products were identified as l-4-(γ,γ-dimethylallyl)tryptophan and inorganic pyrophosphate. DMAT synthetase is a single subunit protein of molecular weight 70,000–73,000 and has an isoelectric point at pH 5.8. The enzyme is activated by Fe²⁺, Mg²⁺, and particularly Ca²⁺; K_m values for l-tryptophan and dimethylallylpyrophosphate were determined to be 0.067 and 0.2 mm, respectively. Kinetic analysis indicated that the DMAT synthetase reaction proceeds by a sequential rather than a ping-pong mechanism.     

Model of growth and ergot alkaloid production by Claviceps purpurea

A growth model for Claviceps purpurea in submerged batch culture is presented. In developing the model, the basic principles of the growth and the morphological properties of C. purpurea are considered. The growth of C. purpurea is assumed to occur in a three-step manner; the first step involves the assimilation and the growth of cells; the second one involves cell division, and the third one involves transformation of the mature cells to a state where they have no ability to divide but do have the ability to produce ergot alkaloids and then they gradually die. Inorganic phosphate is assumed to be the limiting substrate for the first and the second steps in conditions of carbon source being in excess. The model constants are determined by model simulation and graphical searching techniques to find the minimum value of the absolute difference between the experimental and the simulated curves for biomass, alkaloids, and sucrose.     

Glucose catabolism during alkaloid production in a strain ofClaviceps 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. The authors thank Mrs. H. Pechfelder for her skilled technical assistance and Prof. Dr. H. Reinbothe and Dr. C. Wasternack for helpful discussions.