Isolation and characterization of glycocalyx in Streptomyces aureofaciens.

The surface layer, considered to be glycocalyx according to electron-microscopic observations, was separated from a low-production strain of Streptomyces aureofaciens by solubilization with urea and subsequent sonication. The isolation procedure was developed using various agents; neutral phosphatase served as a marker indicating the amount of the material released. The peripheral structure consisted predominantly of glycoprotein and differed from S-layers.

Overproduction of microbial products--facts and ideas.

Overproduction of microbial metabolites is related to developmental phases of microorganisms. Inducers, effectors, inhibitors and various signal molecules play a role in different types of overproduction. Primary and secondary metabolism are interconnected. Biosynthesis of enzymes catalyzing metabolic reactions in microbial cells is controlled by well-known positive and negative mechanisms, e.g. induction, repression, catabolite repression, mechanisms controlling enzyme activity include isosteric and allosteric interactions, e.g. competitive and non-competitive inhibition, allosteric effects, molecular conversion etc. Biosynthesis of secondary metabolites is catalyzed by unaltered enzymes of primary metabolism, by altered enzymes of primary metabolism and by specific enzymes of secondary metabolism. In addition to classical mutagenesis and selection of suitable microbial cells, methods of molecular genetics are used in the overproduction of microbial products.

Subcellular organization of Streptomyces aureofaciens and overproduction of chlortetracycline.

Biosynthesis of chlortetracycline is localized differently under low- and high-production conditions (standard low-production strain and its high-production variant). The experimental evidence was based on the assay of anhydrotetracycline oxygenase in subcellular fractions, ultracytochemical localization and electron-probe X-ray microanalysis of the product in the mycelium. Overproduction of chlortetracycline is closely associated with compartmentation of biosynthetic enzymes and with an efficient export of the antibiotic out of the cell.

A direct-injection reversed-phase liquid chromatographic micromethod for studying the kinetics of terminal reactions of tetracycline biosynthesis.

A new micromethod for measuring enzyme-catalyzed reactions was developed. The method involves a number of consecutive direct injections of aliquots of the reaction mixture onto a microbore column and permits the determination of the time dependence of the decrease of substrate or increase of product concentrations. The reactions proceed in a microvial placed in the autosampler, and as the starting volume can be as low as 10 microliters, the requirement for the amount of enzyme is very low. The autosampler backed by the liquid chromatographic software allows automation of the analyses including data processing and easy quantitation of the enzymatic reaction(s). The method was applied to a system of two consecutive terminal reactions of tetracycline biosynthesis in Streptomyces aureofaciens, catalyzed by anhydrotetracycline oxygenase and tetracycline dehydrogenase. The usage of a diode-array detector facilitated the quantification of the reactions as the product and substrate could be monitored at their optimal wavelengths.

Glucose-6-phosphate dehydrogenase from a tetracycline producing strain of Streptomyces aureofaciens: some properties and regulatory aspects of the enzyme.

Glucose-6-phosphate dehydrogenase from Streptomyces aureofaciens exhibited activity with both NAD and NADP, the maximum reaction rate being 1.6 times higher for NAD-linked activity than for the NADP-linked one. The KM values for NAD-linked activity were 2.5 mM for glucose-6-phosphate and 0.27 mM for NAD, and for NADP-linked activity 0.8 mM for glucose-6-phosphate and 0.08 mM for NADP. NAD- and NADP-linked activities were inhibited by both NADH and NADPH. (2'-phospho-)adenosinediphospho-ribose inhibited only NAD-linked activity. The inhibition was competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate.

Subcellular localization of enzymes in Streptomyces aureofaciens and its alteration by benzyl thiocyanate. I. Phosphatases and ATP-glucokinase.

Mycelia of a low- and a high-production strain of Streptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC 3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2). The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes. Triphosphatase was detected in the periplasmic fraction. Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase. Apyrase was found mainly in the fractions of membranes and cytoplasmic vesicles. Glucokinase was a cytoplasmic enzyme. The enzymes were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 microM) was present in the growth medium.

Subcellular localization of enzymes in Streptomyces aureofaciens and its alteration by benzyl thiocyanate. II. Anhydrotetracycline oxygenase and glucose-6-phosphate dehydrogenase.

The localization of anhydrotetracycline oxygenase and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was studied by determining the enzyme activities in subcellular fractions obtained by differential centrifugation of the mycelia of Streptomyces aureofaciens after lysozyme treatment. Glucose-6-phosphate dehydrogenase was a typical cytoplasmic enzyme both in the low- and high-production strain. Anhydrotetracycline oxygenase was found in the membrane fraction of the low-production strain. In the high-production strain, it was detected in several fractions, the highest activity being found in cytoplasm. The presence of 10 microM benzyl thiocyanate in the culture medium significantly changed the distribution of the latter enzyme in both strains. The redistribution of the enzymes is discussed with respect to tetracycline over-production.

Inhibition studies of glucose-6-phosphate dehydrogenase from tetracycline-producing Streptomyces aureofaciens.

NAD-linked activity of glucose-6-phosphate dehydrogenase from both low-producing and high-producing strains of Streptomyces aureofaciens was inhibited by ATP, ADP, AMP and Pi. The inhibition constants indicate that ADP was the most potent inhibitor. The NADP-linked activity remained unaffected even at relatively high concentrations of these inhibitors. All inhibitions of the NAD-linked activity were competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate. The results represent a possible new regulatory mechanism of glucose-6-phosphate dehydrogenase from a streptomycete and emphasize its involvement in the regulation of the biosynthesis of tetracyclines.

Enzymes of secondary metabolism and the biosynthesis of macrolide antibiotics.

Enzymes participating in the biosynthesis of macrolide antibiotics are reviewed. Enzyme activities are known to play a pivotal role in the formation of biologically active compounds. Hence it is essential to understand these enzymes, their properties and regulation. Macrolide antibiotics represent a relatively compact group of natural products and include several excellent model compounds suitable for enzyme studies that could be generalized to other oligoketide antibiotics.

Effect of inorganic phosphate and benzyl thiocyanate on the activity of anhydrotetracyline oxygenase in Streptomyces aureofaciens.

The level of anhydrotetracyline oxygenase (an enzyme catalyzing the penultimate reaction in the biosynthesis of tetracyline) in Streptomyces aureofaciens was substantially influenced by the amount of inorganic phosphate and by the presence of benzyl thiocyanate in the cultivation medium. Phosphate decreased the specific activity of the enzyme, particularly when added to a growing culture. On the other hand, benzyl thiocyanate increased the specific activity of the enzyme. Its effect was most conspicuous in the growth phase. The effect of benzyl thiocyanate was more pronounced in the low-production strain than in the producing variant. Inorganic phosphate and benzyl thiocyanate did not influence the enzyme activity in vitro. Phosphate added to the growing cultures was readily absorbed by the cells. During this time the enzyme synthesis was repressed, derepresion occurred only after exhaustion of phosphate from the medium. The stimulatory effect of benzyl thiocyanate on the enzyme synthesis was not reversed by the inorganic phosphate added.

Role of phosphatases in the biosynthesis of chlortetracycline in Streptomyces aureofaciens.

ATP diphosphohydrolase activity and inorganic pyrophosphatase reached two maxima during cultivation of the low- and high-producing variant of Streptomyces aureofaciens under conditions of phosphate limitation, i.e. after 30 and 70 h of cultivation. Increased levels of inorganic phosphate in a medium inhibitory to biosynthesis of chlortetracycline markedly decreased the levels of both enzymes. The ATP diphosphohydrolase activity was detected both in the supernatant and membrane fractions of the cell-free preparation of the mycelium.

Properties of apyrase and inorganic pyrophosphatase in Streptomyces aureofaciens.

Apyrase (ATP-diphosphohydrolase, EC 3.6.1.5) and inorganic pyrophosphatase (EC 3.6.1.1) were partially purified from S. aureofaciens RIA 57 and characterized. Apyrase degrades, in addition to ATP, other nucleoside triphosphates and nucleoside diphosphates, diphosphate, thiamine diphosphate, phosphoenolpyruvate and oligophosphates of chain length n less than 90. The apyrase activity was detected in the membrane and supernatant fractions. Its properties (substrate specificity. effect of inhibitors, pH optimum and effect of Mg2+ ions) were similar in both fractions except for the effect of oligomycin that inhibited only the membrane fraction. Pyrophosphatase exhibited a strict substrate specificity, substrates other than diphosphate being degraded relatively slowly. Of other enzymes exhibiting the phosphatase activity acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase (EC 3.6.1.2) and exopolyphosphatase (EC 3.6.1.11) degrading oligophosphatase of chain length n = 15, 40 and 60, were detected.

[Action of chlortetracycline on the enzymes of the producer itself, Streptomyces aureofaciens]. / Deistvie khlortetratsiklina na énzimy sobstvennogo produtsenta Streptomyces aureofaciens.

When preparing a cell-free extract of Streptomyces aureofaciens from the culture to which chlortetracycline (1000 gamma/ml) was added before disintegration with alumina, a considerable decrease in the enzyme activity was reached. The presence of chlortetracycline during disintegration of the cells by means of glass beads did not substantially affect the enzyme activity of the cell-free extract. Addition of chlortetracycline directly to the reaction mixture for the enzyme assay (up to a concentration of 200 gamma/ml) did not induce any inhibition effect. The results suggested that during studying enzyme systems of organisms producing secondary metabolites, the product might distort the results on the use of an inconvenient method.

Synthesis and degradation of proteins and DNA in Streptomyces aureofaciens.

The rate of protein synthesis in Streptomyces aureofaciens, measured by incorporation of U-14C-L-leucine into cells, fluctuated during the production phase in the range of 10-15% of the values determined in the phase of intensive growth. Tetracycline partially inhibited the protein synthesis during the growth phase only. The proteins synthesized between the 6th and 18th hour of growth, were 75% degraded by the 48th hour. The DNA synthesis, measured by means of incorporation of 2-14C-thymine into the mycelium, occurred predominantly during the first 24 h of cultivation. Similarly, DNA synthesized between the 6th and 12th hour of cultivation was degraded by 75% after 48 h. The turnover of culture proteins is thus caused largely by degradation of old cells and growth of new ones which are more resistant to tetracycline. The activity of alanine aminotransferase and aspartate aminotransferase increase substantially towards the end of fermentation.

Intra- and interspecific cosynthetic activity of mutants of Streptomyces coeruleorubidus and Streptomyces galilaeus impaired in the biosynthesis of anthracyclines.

Cosynthesis of anthracycline compounds was followed in five phenotypic groups of mutants of Streptomyces coeruleorubidus (A--E), blocked in the biosynthesis of the daunomycine complex, and in two mutant types of Streptomyces galilaeus (F, G) blocked in the biosynthesis of glycosides of epsilon-pyrromycinone and aklavinone. Glycosides of daunomycinone and 13-dihydrodaunomycinone were produced in combinations A+B, A+C, A+D, A+E and A+F, epsilon-rhodomycinone was synthesized in combinations A+E, A+F, B+E and B+F. During the cultivation of types B--E with type G or F non-anthracycline compounds, typical of S. galilaeus, were cosynthesized. No cosynthesis could be observed in other combinations of the mutant types. Negative results were also obtained with combinations of mutants of the same group and during cultivation of all mutant types with streptomycetes not producing anthracyclines. A scheme illustrating metabolic pathways leading to the biosynthesis of daunomycinone, aklavinone, epsilon-rhodomycinone in S. coeruleorubidus and S. galilaeus was constructed.

Development mutants of Streptomyces coeruleorubidus, a producer of anthracyclines: isolation and preliminary characterization.

The wild strain Streptomyces coeruleorubidus JA 10092 was found to segregate into two spontaneous morphological variants (spo-1 and bld-1) with a different ability to form aerial mycelium in media with glucose as the main carbon source. Six new types of developmental mutants were obtained from the bald variant bld-1 after treatment with mutagens (UV light, gamma radiation, nitrous acid) and after natural selection. Formation of the aerial mycelium was fully suppressed in the bld-2 type growing on media both with glucose and with starch. The other types were bald only on starch media, forming the aerial mycelium on media with glucose; types spo-2, spo-3, spo-4 and spo-5 differed in size, shape and surface structure of spores, the type whi formed asporogenous aerial hyphae.

Improved yields of daunomycinone glycosides in developmental mutants of Streptomyces coeruleorubidus.

When improving Streptomyces coeruleorubidus JA 10092, a producer of antibiotics of the daunomycinone complex, the most active variants were found among isolates of morphological types bld-1 (with a suppressed production of the aerial mycelium on organic media containing glucose) and whi (with an asporogenic aerial mycelium on glucose media and with the bald phenotype on media containing starch). Submerged cultures of the whi mutants produced increased quantities of daunomycinone glycosides in the antibiotic complex, the amount of free anthracyclinones being simultaneously decreased. The whi strains differed from the wild type also in higher demands for aeration, concentration of glucose and in an increased production capacity in starch media. The overall antibiotic activity increased more than 40 times after a six-step selection (application of UV light, gamma-radiation, nitrous acid and natural spreads) combined with an altered fermentation technology.

Mutants of Streptomyces coeruleorubidus impaired in the biosynthesis of daunomycinone glycosides and related metabolites.

Mutants of Streptomyces coeruleorubidus, blocked in the biosynthesis of anthracycline antibiotics of the daunomycine complex, were isolated from the production strains after treatment with UV light, gamma-radiation, nitrous acid, and after natural selection; according to their different biosynthetic activity the mutants were divided into five phenotypic groups. Mutants of two of these groups produced compounds that had not yet been described in Streptomyces coeruleorubidus (aklavinone, 7-deoxyaklavinone, zeta-rhodomycinone and glycosides of epsilon-rhodomycinone). The mutants differed from the parent strains and also mutually in morphological characteristics but no direct correlation between these changes and the biosynthetic activity could be observed in most cases.

Regulation and biosynthesis of secondary metabolites. XVIII. Adenylate level and chlorotetracycline production in Streptomyces aureofaciens.

The relationship was studied between the energy metabolism of the actinomycete Streptomyces aureofaciens and the biosynthesis of chlorotetracycline by this organism. The energy charge values in a culture of low-production strain were almost identical with those of a production variant but the total sum of adenylates was about 10 times higher. In the stationary growth phase both strains evinced a drop in energy charge values followed by a rise to the original level. An increase in the concentration of inorganic phosphate in fermentation medium caused a suppression of antibiotic formation in the lowproduction strain and further rise in the total adenylate level. The expression of the energy charge in Streptomyces aureofaciens acquires a complex character owing to the participation, apart from the adenylate system, of high-molecular polyphosphates as energy donors and the probable lack of a regulating mechanism such as the adenylate kinase reaction.