Oleandomycin content of mycelium and fermentation solution during cultivation of Streptomyces antibioticus

The time-course of the oleandomycin content in the mycelium and fermentation broth-filtrate was studied by the microbiological assay at different periods of cultivation of strains 471 and 961 in fermenters and flasks containing a rich soybean-corn medium. It was shown that centrifugation of the mycelium over the sucrose density gradient induced a 25-80 per cent decrease in its moist weight at the expense of removal of the admixture components of the rich medium. Addition of glucose (2 per cent) to the culture-grown in a lactose medium by the 72nd hour of fermentation had no effect on further increase of the cell biomass. However, it lowered the content of the mycelium-fixed and excreted antibiotic at all the subsequent fermentation periods. The content of oleandomycin in the untreated mycelium was only 0.36 per cent of its content in the fermentation broth filtrate. After centrifugation of the mycelium over the sucrose density gradient and its intensive washing with distilled water the content of the mycelium-fixed antibiotic decreased still more. The time-course of the content of the mycelium-fixed and excreted oleandomycin was characterized by the presence of two activity peaks; by the 80-110th and by the 140-170th hour of cultivation.     

Inhibition of oleandomycin synthesis by glucoses added during fermentation

Oleandomycin biosynthesis by Streptomyces antibioticus is repressed by glucose added to the growth medium in the process of fermentation. Phosphotransferase involved in the synthesis of acetyl CoA and propionyl CoA (the precursors of the antibiotic macrolactone ring) is neither inhibited nor repressed, and the substrate specificity of the enzyme does not change. The content of cAMP in the mycelium of S. antibioticus does not change significantly when either glucose or sucrose is added to the medium 24 h after the inoculation whereas the content of exogenous cAMP rises abruptly 24 h after glucose addition. At the same time, the medium becomes much more acidic and the content of protein in the mycelium rises noticeably. Consequently, cAMP may be involved in the regulation of the culture growth.     

Antibiotic Production of Hyphal Fractions of Streptomyces griseus: II. Streptomycin Production of Different Fractions Obtained by Density Gradient Centrifugation

The streptomycin-producing activity (SPA) of hyphal fractions from washed mycelium of submerged cultures of Streptomyces griseus strain 52-1, as obtained by density gradient centrifugation, was investigated. Activity of the various fractions differed strongly in intensity. The highest SPA was evident in the unfractionated mycelium. A synergistic effect upon SPA was found in the interaction of cultures of different ages, and a 55% increase in yield was obtained by mixing the 48- and 72-hr cultures. A synergistic effect occurred in all combinations studied. By use of fractions obtained from 72-hr mycelium for inoculation, differences in streptomycin production were noted. Some inoculum fractions yielded a greater amount of streptomycin (36%) than the unfractionated mycelial inoculum.     

Kinetic studies on citric acid production by Aspergillus niger. II. The two-stage process.

A two-stage process of submerged citric acid fermentation with replacement of growth medium by fermentation medium has been worked out. The optimum composition of mineral nutrients and pH of the fermentation medium of the second stage of the process were determined. An addition of 0.5 g/l of NH4NO3 as nitrogen source and 0.1 g/l of MgSO4-7H2O as magnesium source ensured effective conversion of sucrose to citric acid. An addition to KH2PO4, on the other hand, was definitely unfavourable as it considerably reduced the product yield. The medium for the second stage of fermentation should be acidified to about pH 2.2, while the water used for washing the mycelium from the remains of the growth medium should have a pH of 2.5--3.5. Under these conditions, with an initial sucrose concentration of 100 g/l, after 132 hr fermentation at 26 degrees up to 90 g/l of citric acid was obtained, which corresponds to a productivity of over 16 g/l. day. The highest activity for citric acid formation was found in three- or four-day-old mycelium.     

Cyclic adenosine-3',5-monophosphate in Streptomyces antibioticus and its possible role in regulating oleandomycin biosynthesis and the growth of the culture

When glucose is substituted for sucrose in the fermentation medium for Streptomyces antibioticus, the pH of the cultural broth becomes more acidic, the rate of protein synthesis in the mycelium rises, and the rate of oleandomycin synthesis decreases abruptly. The dynamics of cAMP (cyclic monophosphate) accumulation was studied in the process of biosynthesis by the culture in different media. Most of the synthesized cAMP (80-90%) was shown to be excreted into the medium. Glucose stimulates cAMP synthesis and excretion from the mycelium by a factor of 1.5-3. No distinct correlation was found between cAMP content in S. antibioticus cells and the level of oleandomycin biosynthesis. A correlation between changes in the concentration of exocellular cAMP and protein synthesis in the mycelium suggests that the excreted cAMP may be involved in regulating the growth of the culture producing the antibiotic.     

Effect of polyploidogenic factors on Trichothecium roseum mycelium in the process of trichothecine and fibrinolytic enzyme biosynthesis

The effect of colchicine and boric acid on the substrate mycelium of Trichothecium roseum was studied in the course of trichothecin biosynthesis. Addition of boric aicd (0.01%) and colchicine (0.1%) to the medium for biosynthesis increased the antibiotic activity of the fungus, this being due to the specific effect of polyploidogenous factors on growth of the mycelium and the proportion of nuclei in it. The number of nuclei increased in cells of the substrate mycelium correlating with a higher antibiotic activity.     

Mannitol Production by Pyrenochaeta terrestris

Pyrenochaeta terrestris (Hansen) Gorenz, J. C. Walker and Larson produces D-mannitol in the mycelium but not in the cutture filtrates when grown in a sucrose salts liquid medium. In the present study, P. terrestris was grown in stilt culture on a synthetic salts medium containing 30 g of sucrose per liter. After inoculation with a myceliat suspension, the mycelial mats were harvested and the dry weight and the amount of mannitol were determined. Maximum mycelial mat production occurred at 15 days after inoculation while the amount of mannitol was greatest at about 7 days after inoculation. The percentage of mannitot on a dry weight basis was maximal (20–25 per cent) within a few days after inoculation and decreased rapidly to 3–4 per cent at the time mycelial mat production was greatest. The same percentage of mannitot was produced when the fungus was grown in shake culture or when the sucrose was replaced by equivalent amounts of D-fructose, D-glucose, D-mannose, maltose, trehalose, and raffinose. Increasing the amount of sucrose or decreasing the amount of sodium nitrate increased the amount of mycelium produced but the percentage of mannitol in the mycelium remained about the same. Mannitot was reutilized when mycelial mats were transferred to a mineral medium without a carbon source. It was concluded that mannitot probably serves as a reserve carbohydrate in P. terrestris.     

Methodological procedures for detecting antibiotic producers among cultures of the family Micromonosporaceae]

Data on intensification of the search for active cultures among Micromonospora are presented. It was shown that the frequency of detecting the antibiotic-producing cultures among Micromonospora under conditions of fermentation on the corn-glucose medium inoculated with agar blocks amounted to 35 per cent. The use of nutrient media of different composition for growing submerged inoculum of Micromonospora demonstrated that the rate of its growth reached maximum on the peastarch medium. The use of this medium for growing submerged seed material for fermentation in the corn-glucose medium increased the frequency of detecting active cultures from 35 to 43.1 per cent. The assay of Micromonospora antibiotic activity twice, i.e. in 96 and 240 hours of the fermentation process increased the frequency of detecting active cultures up to 57.1 per cent and revealing greater variety of antibiotics. Fermentation of Micromonospora cultures simultaneously on 6 different nutrient media inoculated with submerged seed mycelium and assay of the activity for 2 times, i. e. in 96 and 240 hours allowed a detection of up to 76.2 per cent of active strains out of the total number of the isolates.     

Pneumococcal Forssman antigen: enrichment in mesosomal membranes and specific binding to the autolytic enzyme of Streptococcus pneumoniae.

The choline-containing pneumococcal membrane teichoic acid (Forssman antigen) can be isolated with the membrane fractions of the bacteria. The small vesicle (mesosomal) fraction generated during the formation of protoplasts seems to be highly enriched in this material. Forssman antigen was identified in cell fractions on the basis of (i) radioactive choline label, (ii) autolysin-inhibitory activity, and (iii) the sedimentation profile in sucrose density gradients with and without detergent. A membrane teichoic acid could also be isolated from pneumococci grown in medium in which choline was replaced by ethanolamine as the nutritionally required amino alcohol. This material contained radioactive ethanolamine label and behaved similarly to the choline-containing membrane teichoic acid during centrifugation in detergent-containing and detergent-free density gradients. On the other hand, the material had only low autolysin-inhibitory activity. Binding of pure pneumococcal autolysin to micelles of purified Forssman antigen could be demonstrated by mixing these components in vitro and analyzing them by sucrose density gradients and by agarose chromatography. No binding could be observed between the pneumococcal enzyme and the micellar forms of either cardiolipin or polyglycerophosphate-type lipoteichoic acid isolated from Streptococcus lactis.     

Effect of osmotic stress onAspergillus chevalieri respiratory system

The osmotolerant fungusAspergillus chevalieri tolerates up to 80% sucrose concentration in the growth medium. At 50% sucrose the growth rate is 1.5-fold higher than in control (3% sucrose), at 80% sucrose it drops to 30% of the control level. Total proteins and lipids in the mitochondrial fractions obtained from the mycelium rise with increasing sucrose concentration during growth (2.6 and 2.1 times at 80% sucrose). The rate of respiration by whole cells and mitochondrial fractions increases with increased sucrose level in the growth medium. The activity of respiratory enzymes, such as succinate dehydrogenase, cytochrome oxidase, NADH oxidase and succinate oxidase, were also higher in cells growth in the presence of elevated sucrose concentrations. The largest increase was observed with NADH dehydrogenase.A. chevalieri cells grown at high osmotic stress exhibited enlarged mitochondria. The mean mitochondrial diameter at 50 and 80% sucrose was approximately 2.9- and 2.6-fold larger than in the control, respectively. Agarose gel electrophoresis of nucleic acids revealed the presence of high-density bands of RNA in mitochondrial fractions from cells grown at elevated sucrose levels.     

Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.     

Optimization of the medium and cultivation conditions of Penicillium roquefortii f39 producing the diketopiperazine alkaloid roquefortine

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5-2-fold. The increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and amount of the inoculum. The introduction of sucrose into the growth medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.     

Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii: spores, mycelium and yeast cells

To help understand the subcellular machinery responsible for cell wall formation in a fungus, we determined the abundance and subcellular distribution of chitin synthetase (chitin synthase, EC 2.4.1.16) and chitosomes in the asexual life cycle of Mucor rouxii. Cell-free extracts of ungerminated sporangiospores, hyphae/mycelium in exponential and stationary phase, and yeast cells were fractionated by isopycnic centrifugation in sucrose density gradients. The total amount of chitin synthetase per cell increased exponentially during aerobic germination of spores. In all developmental stages, the profile of chitin synthetase activity encompassed a broad range of sucrose density (d = 1.12-1.22) with two distinct zones: a low-density chitosome zone (d = approx. 1.12-1.16) and a high-density, mixed-membrane zone (d = approx. 1.16-1.22). Chitosomes were a major reservoir of chitin synthetase in all stages of the life cycle, including ungerminated spores. Two kinds of chitin synthetase profiles were recognized and correlated with the growth state. In nongrowing cells (ungerminated sporangiospores and stationary-phase mycelium), the profile was skewed toward lower densities with a sharp chitosome peak at d = 1.12-1.13. In actively growing cultures (aerobic mycelium or anaerobic yeast cells), the entire profile of chitin synthetase was displaced toward higher densities; the average buoyant density of chitosomes was higher (d = 1.14-1.16), and more chitin synthetase was associated with denser (d = 1.16-1.23) membrane fractions. In all life cycle stages, chitosomal chitin synthetase was almost completely zymogenic. In contrast to the enzyme from spores or from growing cells, samples of chitosomal chitin synthetase from stationary-phase mycelium were unstable and contained a high proportion of larger vesicles in addition to the typical microvesicles. The presence of chitosomes in ungerminated spores indicates that these cells are poised to begin synthesizing somatic (= vegetative) cell walls at the onset of germination. The increased buoyant density of chitosomes in actively growing cultures suggests that the composition of these microvesicles changes significantly as they mobilize chitin synthetase to the cell surface.     

Short communication: Production of l-glutamate oxidase by Streptomyces sp. N1 in submerged fermentation

In submerged fermentation of Streptomyces sp. N1 in a shake flask, glucose (3% w/v) and (NH₄)₂SO₄ (0.6% w/v) were found to be suitable for extracellular l-glutamate oxidase (GluOx) (EC.1.4.3.11) production. GluOx production was higher with the addition of further KCl or MgCl₂ to the medium within the range of 0 to 0.12% (w/v). The effect of inoculum type, that is, spore inoculation or mycelium inoculation on GluOx biosynthesis was also investigated, and the maximum GluOx production obtained was 2.7 U/ml after 33h fermentation with mycelium inoculation. The results demonstrated a much higher GluOx production and productivity compared with those reported previously.     

嗜线虫致病杆菌产生抗生素的培养基及条件

本对嗜线虫致病杆菌（Xenorhadbus nematophilus）产生抗生素的发酵培养基和发酵条件进行了研究,同时对该菌代谢过程pH值、还原糖、总糖、氨基氮与抗生素产量的关系进行了分析,通过筛选该菌对碳源和氮源的要求,用正交试验初步确定了该菌产素的最佳发酵培养基和条件为：玉米粉1％,大豆粉3％,蔗糖1％,蛋白胨1.5％,KH2PO40.02％,MgSO40.2％,活化剂T0.1％;发酵培养基的起始pH值在6.0－8.0,种龄16h,接种量4％,500mL摇瓶装量15－150mL的条件下培养72h可获得较高的抗生素产量;产素量与菌代谢过程中pH、还原糖、总糖和氨基氮的变化有一定关系,通过培养基和培养条件的研究使该菌的产抗生素能力提高了56.3％。     

Optimization of fermentation condition for antibiotic production by Xenorhabdus nematophila with response surface methodology

Aims: To evaluate the influence of environmental parameters on the production of antibiotics (xenocoumacins and nematophin) by Xenorhabdus nematophila and enhance the antibiotic activity. Methods and Results: Response surface methodology (RSM) was employed to study the effects of five parameters (the initial pH, medium volume in flask, rotary speed, temperature and inoculation volume) on the production of antibiotics in flask cultures by X. nematophila YL001. A 2^5?1‐factorial central composite design was chosen to explain the combined effects of the five parameters and to design a minimum number of experiments. The experimental results and software‐predicted values of production of antibiotics were comparable. The statistical analysis of the results showed that, in the range studied, medium volume in flask, rotary speed, temperature and inoculation volume had a significant effect (P < 0·05) on the production of antibiotics at their individual level, medium volume in flask and rotary speed showed a significant influence at interactive level and were most significant at individual level. The maximum antibiotic activity was achieved at the initial pH 7·64, medium volume in 250 ml flask 25 ml, rotary speed of 220 rev min^?1, temperature 27·8°C and inoculation volume of 15·0%. Maximum antibiotic activity of 331·7 U ml^?1 was achieved under the optimized condition. Conclusions: As far as known, there are no reports of production of antibiotic from X. nematophila by engineering the condition of fermentation using RSM. The results strongly support the use of RSM for fermentation condition optimization. The optimization of the environmental parameters resulted not only in a 43·4% higher antibiotic activity than unoptimized conditions but also in a reduced amount of the experiments. The chosen method of optimization of fermentation condition was efficient, relatively simple and time and material saving. Significance and Impact of the Study: This study should contribute towards improving the antibiotics activity of X. nematophila. Integrated into a broader study of the impact of environmental factors on the production of antibiotic, this work should help to build more rational control strategy, possibly involving scale‐up of production of antibiotics by X. nematophila.     

Production of nikkomycin by immobilized Streptomyces cells — Physiological properties

Summary The antibiotic nikkomycin can be produced by calcium alginate immobilized mycelium of Streptomyces tendae Tü 901 in batch and continuous culture.Scanning electron micrographs show the porous structure of the matrix and the distribution of the cells in the gel.Some physiological properties of free and immobilized mycelia were compared. Immobilization does not change the relative amounts of nikkomycin compounds in the culture broth. DNA and protein content were the same in free and immobilized cells. The specific activity of fructosediphosphate aldolase dropped during fermentation and was lower for entrapped than for free cells. The specific activity of mannitol dehydrogenase increased up to the end of the fermentation and was the same for free and immobilized mycelium.In continuous culture the relative amount of mannitol consumed decreased with increasing flow rate. When the medium was supplemented with amino acids mannitol consumption increased significantly.Dedicated to Prof. Dr. L. Acker on the occasion of his 70th birthday     

Inducing activity of fractionated microsomal material from theXenopus laevis gastrula stage

Summary The postmitochondrial supernatant fromXenopus gastrulae has been fractionated on sucrose gradients. Part of the microsomal material was treated with EDTA, which dissociates most of the polysomal and monosomal material into ribosomal subunits. In addition, a series of pooled fractions from the EDTA treated gradients has been applied to discontinuous gradients in more concentrated sucrose to separate membranous material from the remaining microsomal components.Pooled fractions from all gradients have been tested for inductive activity on amphibian gastrula ectoderm. The spinocaudal (trunk and tail) inducing activity was to some extent eneriched in the membrane fractions.     

Results of the long laboratory storage of Streptoverticillium hachijoense (Jamaguchi) Baldacci, the producer of trichomycin]

Viability, morphologo-cultural features and antibiotic properties of Sv. hachijoense, strain LIA-0052 stored for 10 years in a dry state and in the state of a resting culture were studied. Spores and mycelium of 2-week strains most stable to some chemical and physical factors were used for drying. It was found that viability of strain LIA-0052 was maintained for a longer period of time after lyophilization, in garden soil and agar culture under a layer of mineral oil. By the end of the observation period the viability of the soil culture decreased and the morphologo-cultural properties were stabilized. When the strain was cultivated on media with sucrose, the level of its antibiotic activity increased.     

Rsistance to streptomycin in a producing strain of Streptomyces griseus.

Streptomyces griseus S 104 was sensitive to streptomycin during exponential growth in a medium which, in the subsequent stationary phase, supported production of the antibiotic in yields above 200 mug/ml. When antibiotic production began cultures developed a tolerance toward their lethal metabolite. This was not due to an increase in pH associated with antibiotic production, since pH effects on streptomycin sensitivity in S. griseus were in the reverse direction. However, the degree of tolerance was directly related to the amount of cell material present. Streptomycin production caused no change in the proportion of resistant variants in the population, nor did it cause the severe inhibition of protein synthesis observed in non-producing cultures exposed to the antibiotic. The lack of an effect on protein synthesis is attributed to the absence of streptomycin with in the cytoplasm since soluble extracts from mycelium harvested in the production phase were inactive when bioassayed immediately after cell disruption. However, they developed antibacterial activity rapidly when heated, and more slowly when incubated at 25 degrees C. The addition of phosphatase inhibitors during incubation prevented the appearance of antibiotic activity, and it was concluded that a small amount of streptomycin phosphate is present in the mycelium during antibiotic production. Differences in (14C) streptomycin uptake suggested that the mycelium was appreciably less permeable to the antibiotic in the production phase than during exponential growth. However, a small amount was taken up and much of it was in the soluble fraction of disrupted cells. Bioassays showed that this 14C-labeled antibiotic within the cells had been partially inactivated, suggesting that conversion of streptomycin to an inactive derivative is involved in the mechanism which protects the organism from its metabolite.