Studies on the biosynthesis of the antibiotic crisamicin A and carbon-13 magnetic resonance assignments.

The biosynthesis of crisamicin A, a novel dimeric isochromanequinone antibiotic from Micromonospora purpureochromogenes subsp. halotolerans has been investigated by [1-13C] and [2-13C] labeled acetate precursor feeding experiments. Analysis of the proton noise decoupled and off resonance 13C NMR spectra of 13C enriched and unenriched crisamicin A and their acetate derivatives indicated the biosynthesis via the polyketide pathway, as expected. Further analysis of the enriched spectra allowed the complete assignment of the carbon signals. Of particular interest was the establishment of the linkage between the two monomeric halves of the molecule and determination of the location of the phenolic hydroxyls.

Crisamicin A, a new antibiotic from Micromonospora. I. Taxonomy of the producing strain, fermentation, isolation, physico-chemical characterization and antimicrobial properties.

A microorganism, designated as RV-79-9-101 and now identified as Micromonospora purpureochromogenes subsp. halotolerans, isolated from a mud sample in the Philippines, has been shown to produce a complex of antibiotics called crisamicins. Thin-layer chromatography and bioautography, employing solvent extracts of whole fermentation broths, revealed a minimum of five antimicrobial components. The major biologically-active component of the antibiotic complex, crisamicin A, was obtained in pure form after preparative silica gel column chromatography followed by crystallization. Based on physico-chemical data crisamicin A has been identified as a novel member of the isochromanequinone group of antibiotics. It exhibits excellent in vitro activity against Gram-positive bacteria but little or no activity towards Gram-negative bacteria or fungi.

Effect of phosphate and copper on the fermentation of hydroheptin.

The organism Streptomyces chartreusis IMRU 3962 produced a mixture of the antibiotics hydroheptin and chartreusin in fermentation broth. The addition of increasing levels of phosphate resulted in a corresponding increase in the production of both antibiotics, with maximum yields of 400 to 450 micrograms of chartreusin per ml and 80 to 100 micrograms of hydroheptin per ml at 0.45 to 0.55 M phosphate. Chartreusin was invariably produced at a higher ratio; however, a reversal in ratio to favor hydroheptin was attained when 0.03% copper sulfate was added to the medium, particularly at a 0.2 M KH2PO4 level, with antibiotic yields of 125 micrograms of hydroheptin per ml and 40 micrograms of chartreusin per ml.

Comparative analysis of hexaene antibiotics.

A study of nine hexaene antibiotics resulted in their assignment to three subgroups on the basis of their bioactivities. Separation of individual components of the nine antibiotic complexes was accomplished by thin-layer chromatography. Similarities and differences among members of the subgroups were established by thin-layer chromatography, spectrophotometry and high performance liquid chromatography. Two antibiotics (endomycin and hexafungin) were found to be similar.

Hydroheptin: a water-soluble polyene macrolide. I. Taxonomy, fermentation and isolation.

A soil isolate of Streptomyces, which has been deposited in the culture collection of the Waksman Institute of Microbiology, Rutgers University as IMRU 3962, produces a new heptaene macrolide antifungal antibiotic, hydroheptin. The producing microorganism, which co-produces the antibiotic, chartreusin, has been identified as a strain of Streptomyces chartreusis. Fermentation and bioassay procedures were developed for the production and analysis of hydroheptin and chartreusin. Isolation and purification procedures based on solvent extraction and precipitation of an organic acid yielded a relatively pure product of hydroheptin.

Hydroheptin: a water-soluble polyene macrolide. II. Chemical and biological properties.

Hydroheptin, a new polyene macrolide antifungal antibiotic, is co-produced with the antibiotic, chartreusin, by a strain of Streptomyces chartreusis designated as IMRU 3962 isolated in our laboratory. The unique water-solubility of this antibiotic at neutrality, revealing in aqueous solution molecular dispersion and an ultraviolet-visible absorption spectrum characteristic of an all-trans heptaene chromophore, clearly distinguishes it from all previously-described and naturally-occurring heptaene macrolides. The isolation and identification of the amino sugar, mycosamine (3-amino-3,6-dideoxy-D-mannose), in acid hydrolysates of hydroheptin and the absence of an aromatic amine upon retrograde alkaline dealdolization of the molecule certainly characterize the antibiotic as a member of the non-aromatic heptaene macrolide group. Chromatographic and countercurrent distribution studies likewise support its novelty. With little or no demonstrable activity against bacteria, hydroheptin as compared to other non-aromatic heptaene macrolides exhibits excellent but somewhat less activity against a wide variety of yeasts and fungi. Likewise, its parenteral toxicity appears to be less than that of other heptaene macrolides.

New antibiotic pigments related to fusarubin from Fusarium solani (Mart.) Sacc. I. Fermentation, isolation, and antimicrobial activities.

A cholesterol-decomposing fungus, Fusarium solani (Mart.) SACC. strain PP 96, was found to produce several different naphthaquinone pigments in a glycerol-mineral salts medium. Three novel compounds structurally related to fusarubin were isolated by chloroform extration followed by silicic acid column chromatography and preparative thin-layer chromatography. The purified compounds were found to have relatively low activity against bacteria, yeasts and filamentous fungi.

Specific inhibition of candicidin biosynthesis by the lipogenic inhibitor cerulenin.

Cerulenin, an inhibitor of fatty acid synthesis, inhibits specifically the biosynthesis of the polyene macrolide candicidin by resting cells of Streptomyces. 50% inhibition was achieved with a cerulenin concentration of 1.5 mug/ml. Cells in which candicidin synthesis was inhibited for 10 h remained capable of candicidin synthesis after removal of the inhibitor. Cerulenin inhibits specifically the incorporation of [14C] propionate into candicidin but does not affect total protein or RNA synthesis. The uptake of [14C] propionate was not inhibited under conditions which totally prevented the incorporation of propionate into candicidin. Incorporation of p-amino[14C] benzoic acid NH2 [14C] BzO- into the aromatic moiety of candicidin was also inhibited by cerulenin. The inhibitory action of cerulenin was not reversed by exogenous fatty acids. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, it is concluded that the polyene macrolide candicidin is synthesized via the polyketide pathway by condensation steps similar to those occurring in fatty acid biosynthesis.

Candihexin polyene macrolide complex: physicochemical characterization and antifungal activities of the single components.

The candihexin complex produced by a natural mutant of Streptomyces viridoflavus has been resolved into eight components. At least six of them, having hexaene chromophores, can be separated directly by thin-layer chromatography. The four major components, candihexins A, B, E, and F, account for about 90% of the total complex. They have been isolated and purified to homogeneity. All the major components were found to have macrolide lactone rings with all-trans hexaene chromophores. Candihexins A and B have compositions and molecular weights similar to candidin, the heptaene polyene macrolide produced by the parent S. viridoflavus, whereas candihexins E and F show smaller molecular weights and rather different elementary compositions. Candihexins A and B yield the amino sugar mycosamine upon hydrolysis, but no sugar was found in hydrolysates of candihexins E and F. Candihexins A and B have high antifungal activity against yeasts and filamentous fungi. The amino sugar-lacking candihexins E and F show no antifungal activity at levels of 100 mug/ml.

Sequence of biosynthesis of the components of the polyene macrolides candidin and candihexin: macrolide aglycones as intracellular components.

A sequential formation of the single components of the polyene macrolide candidin complex (heptaene) has been found. In addition to the three components occurring in the candidin complex at the end of the fermentation, two other "early" all-trans heptaene components have been characterized. They exist only during the phase of active biosynthesis of candidin. Two of the components of the polyene macrolide candihexin complex (hexaene) that have been described as lacking amino sugar were the only intracellular (mycelium-associated) components observed under conditions in which no extracellular polyene remained attached to the producing cell. The results indicate that glycosylation of the macrolide ring takes place during the secretion process.

Factors affecting the production of candicidin.

Factors affecting candicidin synthesis and mycelial growth of Streptomyces griseus IMRU 3570 were studied. Inorganic phosphate was found to inhibit candicidin synthesis but to stimulate mycelial growth. Zinc, iron, and magnesium ions stimulated candicidin synthesis at relatively high concentrations in a complex medium but not in a synthetic medium. No other factors studied, such as temperature, oxygen absorption rate, and sugar concentration, were found to differentially affect antibiotic synthesis and mycelial growth. Optimum concentration of inorganic phosphate for candicidin synthesis in a chemically defined medium was found to be between 5 x 10(-5) and 5 x 10(-4) M. The culture in idiophase stage can be reverted to typical trophophase growth by the addition of inorganic phosphate, suggesting the controlling role of inorganic phosphate in repression and derepression of secondary metabolic and primary metabolic activity of the culture. With a soya peptone-glucose medium, the maximum rate of candicidin production could be maintained and extended for a considerable length of time by controlling the culture pH at 8.0, using glucose to adjust the pH during the later stages of a batch fermentation. Carrying out fermentations in this way has given candicidin yields up to 4 g/liter.

Quantitative thin-layer spectrodensitometric determination of the components of polyene macrolide antibiotice complexes.

A direct spectrodensitometric method for quantitating the components of polyene macrolide complexes after separation by thin-layer chromatography is described. Resolution of the components of the candidin and candihexin complexes was good up to 2.5 and 10 mu-g/spot, respectively. The peak areas were linear with the amount spotted up to the same levels. Maximum peak areas for the components of the candidin and candihexin complexes were obtained using light wavelengths of 360 and 340 nm, respectively. Spotting errors rather than instrumental parameters were responsible for the variance of repeated determinations. Minimal relative standard deviation values were found at intermediate concentration levels in the linear range.

Purification of phosphomannanase and its action on the yeast cell wall.

An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.

Effect of shaking speed and type of closure on shake flask cultures.

Growth of microorganisms and biosynthesis of microbial products in shake flasks may be limited by operating conditions which provide inadequate supplies of oxygen. Methods are described for meeting the oxygen requirements of test organisms by using standard baffled flasks with pad-type closures and shaking at relatively high speeds. Growth of Escherichia coli B in a rich medium and production of candidin by Streptomyces viridoflavus were the test systems used. Flasks shaken at 230 to 385 rev/min gave sulfite oxidation rates of 1 to 8 mmoles of oxygen per liter per min over a useful working volume range (40 to 150 ml in 300-ml flasks). These rates are as high as those obtained in agitated fermentors under usual operating conditions.

Liquid nitrogen preservation of standard inoculum: gas-phase storage.

Liquid nitrogen storage was the most satisfactory of several methods tested for supplying standard Streptomyces viridoflavus inoculum for laboratory and pilot plant experimentation. Shake-flask cultures were subdivided into sterile cotton-plugged ampoules and stored in the gas space of a liquid nitrogen refrigerator. There were no detectable changes in viability or in candidin-producing capacity over a 12-month test period. The procedure also proved satisfactory with all other organisms tested.