Intracellular cyclic adenosine 3',5'-monophosphate levels and streptomycin production in cultures of Streptomyces griseus.

Changes in the amount of cyclic 3',5'-adenosine monophosphate within the mycelium of Streptomyces griseus were measured as cultures progressed through trophophase and idiophase in a complex medium supporting growth and streptomycin synthesis. Concentrations were highest before the cultures entered stationary phase and had declined 90% by 5 h before the antibiotic was produced. This low conentration was maintained while the antibiotic accumulated during the idiophase. The results indicate that the onset of streptomycin synthesis is not directly mediated by an increase in intracellular cyclic 3',5'-adenosine monophosphate concentration, and thus that antibiotic production in S. griseus is not controlled by catabolite repression.     

Constitutive Expression of Enniatin Synthetase during Fermentative Growth of Fusarium scirpi

The production of enniatins by Fusarium scirpi during fermentative growth in submerged cultures was measured. The fungus produced the antibiotic during mycelial growth, but not during the stationary phase of cultivation. By contrast, enniatin synthetase, the enzyme responsible for enniatin synthesis, was present during growth, during the stationary phase, and even in spores. Similarly, the enniatin synthetase mRNA was present at every stage of the cultivation of the fungus. Therefore, this multifunctional peptide synthetase is a constitutive enzyme, the expression of which is not regulated by any specific mechanism. The findings stand in contrast to the common assumption that production of secondary metabolites underlies regulatory control, leading to separation of the trophophase and the idiophase.     

Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin

The effect of ammonium ions on growth and tylosin biosynthesis in Streptomyces fradiae NRRL 2702 cultured on a chemically defined medium was studied. Mycelial growth and tylosin production were not affected when ammonium sulphate was added to idiophase cultures to a final concentration of 10 mm or 20 mm; however, when ammonium sulphate was added to tylosin cultures to a final concentration of 20 mm before the onset of antibiotic biosynthesis (trophophase), tylosin production was severely suppressed while mycelial growth was stimulated. The activities of propionyl-coenzyme A carboxylase (EC 6.4.1.3) and methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1), enzymes involved in the synthesis of tylonolide precursors, were depressed in high ammonium cultures. The activity of macrocin 3′-o-methyltransferase, which catalyses the methylation of macrocin to form tylosin, was also affected by high concentrations of ammonium ions added in the trophophase.     

Oxidative state in idiophase links reactive oxygen species (ROS) and lovastatin biosynthesis: Differences and similarities in submerged- and solid-state fermentations

The present work was focused on finding a relationship between reactive oxygen species (ROS) and lovastatin biosynthesis (secondary metabolism) in Aspergillus terreus. In addition, an effort was made to find differences in accumulation and control of ROS in submerged (SmF) and solid-state fermentation (SSF), which could help explain higher metabolite production in the latter. sod1 expression, ROS content, and redox balance kinetics were measured during SmF and SSF. Results showed that A. terreus sod1 gene (oxidative stress defence enzyme) was intensely expressed during rapid growth phase (trophophase) of lovastatin fermentations. This high expression decreased abruptly, just before the onset of production (idiophase). However, ROS measurements detected high concentrations only in idiophase, suggesting a link between ROS and lovastatin biosynthesis. Apparently sod1 down regulation promotes the rise of ROS during idiophase. This oxidative state in idiophase was further supported by a high redox balance observed in trophophase that changed to a low value in idiophase (around six-fold lower). The patterns of ROS accumulation, sod1 expression, and redox balance behaviour were similar in SmF and SSF. However, sod1 expression and ROS concentration (ten-fold), were higher in SmF. Our results indicate a link between ROS and lovastatin biosynthesis. Also, showed differences of physiology in SSF that yield lower but more steady ROS concentrations, which could be associated to higher lovastatin production.     

Monitoring microbial product synthesis by measurement of the dehydrogenase activity

A method for the photometric measurement of dehydrogenase activities with the redox dye phenazine methosulphate at a fixed biomass concentration was applied for the investigation of idiophasic product synthesis. It was found that in citric acid fermentation by Yarrowia lipolytica which is characterized by a well defined separation into tropho-and idiophase the magnitude of product synthesis rates corresponds to the level of dehydrogenase activity at the end of the trophophase. In cellulase production by Trichoderma reesei the tropho-and idiophase are not well defined but a rise of dehydrogenase activity in the course of the idiophase is likely to correspond to an increase of the cellulase formating rate. The significance of the relation of dehydrogenase activity to production rates for monitoring industrial fermentations is discussed.     

Production of the macrolide antibiotic tylosin in batch and chemostat cultures

The production of tylosin and related compounds by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture, a trophophase–idiophase kinetic pattern was observed with tylosin, macrocin, and relomycin accumulating in the idiophase. When the organism was grown in chemostat culture, the specific rate of production of tylosin and related compounds (q_tylosin) was found to be a function of the growth rate. The maximum value of (q_tylosin) was observed when D = 0.017 hr^?1. At this growth rate only tylosin and relomycin accumulated in the medium. By varying the concentration of glucose in the ingoing medium it was possible to study the effects of glucose on tylosin synthesis in chemostat cultures. At a growth rate of 0.017 hr^?1, the maximum value of q_tylosin was 0.71 mg tylosin/g dry weight (DW)/hr when the glucose uptake rate was 7 mg glucose/g DW-hr. This value of q_tylosin was 40% greater than the maximum q_tylosin observed in batch culture. When glycerol was substituted for glucose in the medium, it was possible in chemostat culutures to get values of q_tylosin approximately 20% greater than those obtained with glucose at the same uptake rate. By varying the concentration of sodium glutamate in the ingoing medium it was possible to show that increasing the specific uptake rate of sodium glutamate increased the values of q_tylosin obtained. Similar chemostat experiments where the inorganic phosphate concentration in the ingoing medium was varied showed that increased the uptake of phosphate decreased the values of q_tylosin obtained. Also increasing the uptake rate of phosphate increased the relomycin-to-tylosin ratio. By taking into consideration the suppressing effects of glucose and the stimulating effects of sodium glutamate on tylosin synthesis, it was possible to formulate a medium that resulted in a value of q_tylosin of 1.1 mg/g/hr being obtained at a growth rate of 0.03 hr^?1. Batch fermentations with this medium did not follow a trophophase–idiophase kinetic pattern, but instead tylosin was actively synthesized during a period of rapid mycelial growth.     

Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus

The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.     

Cephamycin production and isopenicillin N synthetase activity in cultures of Streptomyces clavuligerus

Summary The relationships between growth, cephamycin production and isopenicillin N synthetase (IPNS) activity in cultures of Streptomyces clavuligerus were examined to establish conditions that optimize the yield and specific activity of the enzyme. Unexpectedly for a secondary metabolic pathway component, IPNS was synthesized preferentially during rapid growth and reached its maximum specific activity in cultures supplied with readily assimilated sources of nitrogen. The activity decreased sharply as cultures entered stationary phase. On the other hand, comparisons of growth and antibiotic production on a range of carbon and nitrogen sources as well as measurements of IPNS activity in chemostat cultures implicated catabolite repression, a mechanism usually associated with separation of trophophase and idiophase activities, as an important factor in controlling expression of the secondary metabolic pathway. An explanation for the timing of IPNS biosynthesis is suggested.Dedicated to Professor H. J. Rehm on the occasion of his 60th birthday     

Role of the carbon source in regulating chloramphenicol production by Streptomyces venezuelae: studies in batch and continuous cultures

Both carbon- and nitrogen-limited media that supported a biphasic pattern of growth and chloramphenicol biosynthesis were devised for batch cultures of Streptomyces venezuelae. Where onset of the idiophase was associated with nitrogen depletion, a sharp peak of arylamine synthetase activity coincided with the onset of antibiotic production. The specific activity of the enzyme was highest when the carbon source in the medium was also near depletion at the trophophase-idiophase boundary. In media providing a substantial excess of carbon source through the idiophase, the peak specific activity was reduced by 75%, although the timing of enzyme synthesis was unaltered. Moreover, chemostat cultures in which the growth rate was limited by the glucose concentration in the input medium failed to show a decrease in specific production of chloramphenicol as the steady-state intracellular glucose concentration was increased. The results suggest that a form of "carbon catabolite repression" regulates synthesis of chloramphenicol biosynthetic enzymes during a trophophase-idiophase transition induced by nitrogen starvation. However, this regulatory mechanism does not establish the timing of antibiotic biosynthesis and does not function during nitrogen-sufficient growth in the presence of excess glucose.     

Regulation of citric acid production by oxygen: Effect of dissolved oxygen tension on adenylate levels and respiration in Aspergillus niger

Summary The mechanism of the control of citric acid accumulation by oxygen was investigated by means of pilot plant fermentation using Aspergillus niger. The critical dissolved oxygen tension (DOT) for oxygen uptake of this fungus was about 18–21 and 23–26 mbar for trophophase and idiophase, respectively. Minimal DOT for citric acid production was about 25 mbar. Citric acid production increased steadily between 40–150 mbar. Short time changes in the DOT produced immediate, irreversible changes in the rate of product formation. Adenine nucleotides paralleled growth but showed no evidence for control function in the oxygen effect on citric acid fermentation. A branched respiratory system was identified by experiments using specific inhibitors (antimycin, cyanide, azide, rotenone, amytal and salicylhydroxamic acid). Growth was sensitive towards inhibitors of the standard respiratory chain, but only slightly sensitive towards salicylhydroxamic acid (SHAM). Citric acid synthesis was highly sensitive towards SHAM during trophophase, but sensitive towards antimycine during idiophase. Interruptions in aeration cause an impairment of the SHAM sensitive oxidase during trophophase, and of the antimycin sensitive oxidase during idiophase.Dedicated to emeritus Professor Dr. Richard Brunner on the occasion of his 80th birthday     

Kinetics of biosynthesis of polyene macrolide antibiotics in batch cultures: Cell maturation time

The changes in several parameters of the candicidin fermentation (total, mycelium-associated, and extracellular product formation; growth rate; DNA content; glucose utilization; dissolved oxygen in the broth; and oxygen uptake rate) during the trophophase-idiophase transition are compared with previously reported data for the polyene macrolides, candidin and candihexin. The maturation time, t_m, and the productformation rate constant, k_p, have been calculated for each of the three polyene macrolides using a simple mathematical model. Slow-feeding of glucose, which resulted in candidin and candihexin overproduction has been shown to increase the polyene formation rate constant and toretard trophophase to idiophase transition (longer maturation time). The opposite effect is achieved by repeated feeding of soybean meal. The values of the maturation times and polyene formation rates obtained were used to predict the production of polyene macrolide antibiotics in batch cultures.     

Effect of Sclerin on Production of the Aminoglycoside Antibiotics Accompanied by Salvage Function in Streptomyces

Sclerin (SCL) stimulated the production of aminoglycoside antibiotics. Production of kanamycin (KM) was preceded by formation of KM-N-acetyltransferase and initiated by induction of N-acetyl-KM-amidohydrolase. KM-acetyltransferase rapidly developed and suddenly decreased at an early trophophase, whereas N-acetyl-KM-amidohydrolase appeared late and increased gradually. Addition of SCL to the culture initially most enhanced the productivity of KM, inducing both enzymes. Production of ribostamycin (RM) was also preceded by RM-acetyltransferase and optimal period for SCL addition was initial. On the other hand, production of streptomycin (SM) associated with both SM-(streptidino) kinase and alkaline phosphatase through trophophase and idiophase was rather stimulated by SCL added later. SCL induced alkaline phosphatase but not SM-(streptidino) kinase. Thus, a difference has been found in the effect of SCL between regulation of aminoglycoside-modifying (salvaging) enzymes and productivity of antibiotics.     

Indirect estimation of biomass by rapid ribonucleic acid determination

Summary Microbial ribonucleic acid (RNA) was estimated by determinations of nucleotides formed by complete hydrolysis. The steps involved in this procedure comprise the alkaline hydrolysis of RNA (5 N NaOH; 100°C; 5 min) to a mixture of 2-and 3-mononucleotides and successive separation and quantification by means of high performance liquid chromatography (HPLC). Since guanylic acid (GMP) shows adequate stability during RNA degradation and may be separated well and rapidly from other compunds, it is used as an equivalent measure of RNA. The assay is performed within 15 min having a lowest detection limit of 4 mg RNA/l. It was applied to isolated yeast cells and fermentation broths of mycelial organisms. Linear correlation to mycelial dry weight was established for unlimited growth (trophophase), while in idiophase determination of RNA may serve as a measure of active biomass.     

Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.     

The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Studies on Scenedesmus acutus growth II. Effect of autotrophic and mixotrophic growth on the amino acid and the carbohydrate composition of Scenedesmus acutus

As a general rule an increase in carbohydrates occurs during the light phase of the cell cycle and that of protein during phase, although variations were found in these components under autotrophic and mixotrophic growth conditions. The results are based on the quantitative determination of carvohydrates as trimethylsilyl (TMS) derivatives and amino acids as N-trifluoroacetyl-n-butyl (TAB) esters in algal cells cultured in light and dark periods by gas-liquid chromatography (LC). Cells harvested during the dark period contained more amino acids as compared to similar cultures harvested during the light phase. In light, the production of amino acids of the aspartate family increased in cells cultivated with glucose and carbon dioxide. With glucose as sole carbon source, the carbohydrate content was higher in the dark than in the light period. Under continuous light conditions, in the presence of carbon dioxide, there was a decrease in the carbohydrate content also. Gas-liquid chromatography analysis of the extract of the purified cell walls showed that they are made up of 0.076% carbohydrates and 0.28% amino acids on the dry weight (DW) basis of whole cells. The results on the metabolism of cells, under autotrophic and mixotrophic conditions, are discussed in this article.     

Improved production of the anticancer compound 1403C by glucose pulse feeding of marine Halorosellinia sp. (No. 1403) in submerged culture

Effects of different pulse fed-batch methods on production of the anti-cancer compound 1403C by marine mangrove endophytic fungus Halorosellinia sp. (No. 1403) in a 5-L bioreactor were investigated. Since high glucose concentrations improved mycelial growth but inhibited 1403C production, the cultures were pulse fed with glucose solutions to keep the residual glucose lower than 4 g/L but higher than 0.5 g/L during rapid growth phase (0-50 h). In this way, a maximum dry biomass, 1403C production and yield coefficient (Y1403C/X) of up to 4.5 g/L, 2.64 g/L and 0.59 g/g dry cell weight, respectively were achieved. These values are 22.7%, 98.0% and 61.4%, respectively higher than those obtained with batch cultures. This strategy is valuable for fermentation scale-up of Halorosellinia sp. (No. 1403) for 1403C production, and might also be applicable to other marine fungi cultures.     

Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene

One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work we have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. We show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h. g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate (51 mg/h. g dry cell weight). The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting.