Biosynthesis of nanaomycin. II. Purification and properties of nanaomycin D reductase involved in the formation of nanaomycin A from nanaomycin D1.

Nanaomycin D reductase, catalyzing the conversion of nanaomycin D to nanaomycin A, which is the first step in the biosynthetic sequence (D leads to A leads to E leads to B) in Streptomyces rosa var. notoensis, was purified from the crude extract of the strain by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite to give an electrophoretically homogeneous preparation. The enzyme was found to be a flavoprotein which contains FAD as a prosthetic group and has a molecular weight of 68,000 daltons. It catalyzed the reductive transformation of nanaomycin D to nanaomycin A in the presence of NADH under anaerobic conditions. The Km values were 250 microM for nanaomycin D and 62 microM for NADH. The enzyme was inhibited by 1 mM Cu2+ ion and by NADH at concentrations over 50 microM. The optimal pH was 5.0 and the optimal temperature was 37 degrees C. Several benzoisochromane-quinone antibiotics other than nanaomycin D, kalafungin (enantiomer of nanaomycin D), griseucin A and frenolicin B were converted to the corresponding reduced products by the enzyme. However, granaticin and 4 alpha, 10 alpha-epoxynanaomycin D were not converted.

Biosynthesis of nanaomycin: syntheses of nanaomycin E from nanaomycin A and of nanaomycin B from nanaomycin E in a cell-free system.

The enzyme reactions from NNM-A to NNM-E and from NNM-E to NNM-B were established in a cell-free system containing an extract from the nanaomycin producer Streptomyces rosa var. notoensis. The enzyme which catalyses the former epoxide-forming reaction (NNM-A leads to NNM-E) required NADH (or NADPH) and O2 suggesting that it is the monooxygenase-type; thus, NNM-A monooxygenase (4a,10a-epoxidizing) is proposed as its name. The other enzyme which catalyses the reductive epoxide-opening reaction (NNM-E leads to NNM-B) requires NADH or NADPH and is tentatively named NNM-B synthetase. Such a reductive epoxide-opening reaction is a novel enzyme reaction.

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.

Control of ammonium ion level for efficient nanaomycin production.

The addition of a small amount of NH4+ to a complex medium increased nanaomycin production by Streptomyces rosa subsp. notoensis OS-3966. The best NH4+ donor for nanaomycin production was NH4+-saturated natural zeolite, with which the maximum titer of nanaomycin E was 760 micrograms/ml, about four fold higher than the control titer. In contrast, lowering NH4+ levels by adding NH4+-trapping agents such as untreated natural zeolite reduced antibiotic production.

Studies on a new antibiotic M-92 produced by Micromonospora. I. Taxonomy of M-92 producing Micromonospora and antibiotic production therefrom.

An isolate (strain MCRL 0404) producing a new antibiotic, M-92, was identified as a new strain of Micromonospora for which the name Micromonospora verruculosa sp. nov. was proposed. A water-infusion of dried sea tangle and dried shiitake was utilized for the production of M-92. When this strain was fermented in the medium containing this infusion, M-92 accumulated in the mycelium at about 10 times that in the broth at the peak level.

Studies on a new antibiotic M-92 produced by Micromonospora. IV. Bactericidal action of the component VA-2.

The action of VA-2, the most active component of antibiotic M-92, against S. aureus is bactericidal but not bacteriolytic. The bactericidal action is markedly affected by incubation temperature, whether bacterial cells are prolific or resting. The bactericidal kinetics of VA-2 is biphasic, since addition of VA-2 caused rapid and straight decrease in viability curve and reached a plateau after several minutes. The bactericidal activity of VA-2 is blocked by 2,4-dinitrophenol. Alike to many membrane-active bacteriocins, VA-2 seems to exert its action through two stages.

New anthracyclinone metabolites from two blocked mutants of Streptomyces galilaeus MA144-M1.

Two blocked mutants of the aclacinomycin-producing Streptomyces galilaeus MA144-M1 produced new anthraquinones and anthracyclinones. The mutant ANR-58 produced compounds 58A, 58B, 58C (7-deoxy-2-hydroxyaklavinone), 58D (2-hydroxyaklavinone) and 58WR. All these compounds have the 2-hydroxyl group. The mutant ANR-665 produced compounds 665A and 665B. The compounds 58A, 58B and 665A have an anthraquinone skeleton.

Biosynthesis of nanaomycin. III. Nanaomycin A formation from nanaomycin D by nanaomycin D reductase via a hydroquinone.

Nanaomycin D reductase which is involved in the biosynthesis of the antifungal antibiotic nanaomycin catalyzes the formation of nanaomycin A from nanaomycin D in the presence of NADH under anaerobic conditions. On the other hand, under aerobic conditions NADH is consumed and nanaomycin A formation is markedly reduced. These findings suggest that nanaomycin A synthesis is not due to the direct reduction of the 5-membered lactone ring of nanaomycin D. Reduction of various quinones by the enzyme was examined. It was found that nanaomycin A is converted to its hydroquinone derivative in the presence of NADH under anaerobic conditions, whereas NADH consumption alone is observed under aerobic conditions. When p-benzoquinone, 1,4-naphthoquinone or menadione is used instead of nanaomycin D, NADH is also consumed. These results indicate that: (1) these compounds act as electron acceptors, (2) O2 functions as final electron acceptor under aerobic conditions, and (3) nanaomycin D reductase is, in fact, an NADH dehydrogenase (quinone). Changes in the UV-absorption spectrum of a reaction mixture containing nanaomycin D and NADH indicate that a hydroquinone derivative is formed as an intermediate during nanaomycin A formation. Similar results were obtained when nanaomycin D is reduced chemically with NaBH4 or Zn powder. It was concluded that nanaomycin D is converted to a hydroquinone derivative and that nanaomycin A is then formed nonenzymatically through intramolecular electron transfer.

Biosynthesis of napyradiomycins.

Biosynthetic studies on napyradiomycins were carried out based on the incorporation of [2-13C]acetate and [1,2-13C]acetate. The alignment of acetate units suggested that the B and C rings of napyradiomycins are derived from a pentaketide, while ring A and the side chain may be synthesized from mevalonate.

Biosynthesis of the antibiotic actinorhodin. Analysis of blocked mutants of Streptomyces coelicolor.

From two types of class V act mutants of Streptomyces coelicolor two monomeric precursors of actinorhodin have been isolated and their structures determined. One is the known antibiotic kalafungin and the other a new compound. Their relationship to actinorhodin biosynthesis is discussed.

Isolation and structure elucidation of a novel griseorhodin.

Three antibiotics possessing cytotoxic properties were isolated from a strain of Streptomyces griseus (FCRC-57). One was found to be identical with griseorhodin A. A second, FCRC-57-U, was found to be identical to griseorhodin C. FCRC-57-G is a new antibiotic structurally related to griseorhodins A and C, and is active against KB cells in vitro. The structure of this new antibiotic was determined using mass spectrometry, proton and carbon nuclear magnetic resonance spectroscopy and synthesis.

Sarubicin b, a new quinone antibiotic, isolated from the fermentation broth of a streptomyces strain.

Sarubicin B, isolated from the culture filtrate of a Streptomyces strain JA 2861, is a new quinone antibiotic. The compound was isolated as an orange crystalline powder, mp 282 approximately 284 degrees C. In vitro sarubicin B was found to inhibit Gram-positive bacteria. It was not active against Gram-negative microorganisms.

Bioconversion and biosynthesis of nanaomycins using cerulenin, a specific inhibitor of fatty acid and polyketide biosyntheses.

The biosynthetic relationship of the nanaomycins produced by Streptomyces rosa var. notoensis OS-3966 was studied by means of a bioconversion method using the antibiotic cerulenin, a specific inhibitor of fatty acid and polyketide biosyntheses. Nanaomycin D was considered to be the first component produced from the hypothetical intermediate "polyketide". It is proposed that the biosynthesis sequence for the nanaomycin is: nanaomycin D leads to nanaomycin A leads to nanaomycin E leads to nanaomycin B. Nanaomycin B can be converted to nanaomycin A by non-enzymatic dehydration; however, nanaomycin A is rapidly bioconverted to nanaomycin E, which is the major component synthesized by the nanaomycin-producing strain.

Studies on new antibiotics SF2415. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

A new species of Streptomyces is described for which the name Streptomyces aculeolatus is proposed. The organism produces new antibiotics SF2415A1, A2, A3, B1, B2 and B3 active against Gram-positive bacteria. Empirical molecular formulae of the antibiotics SF2415A1, A2, A3, B1, B2 and B3 were determined to be C26H31N2O5Cl, C26H30N2O5, C26H30N2O5Cl2, C26H33O5Cl, C26H32O5 and C26H32O5Cl2, respectively.

Crisamicin C, a new isochromanequinone antibiotic. Isolation, structure determination, and biosynthesis.

Micromonospora purpureochromogenes subsp. halotolerans was found to produce crisamicin C, a novel antibiotic, together with crisamicin A. Crisamicin C was purified by silica gel column chromatography and its physico-chemical properties, structure and biosynthesis were studied. Crisamicin C, mp 260 degrees C (dec), showed UV maxima at 392 (epsilon 9,497), 261 (epsilon 32,959) and 232 nm (epsilon 24,623) in CH3CN, and gave an IR spectrum with absorbances at 1782 (lactone), 1705 and 1655 (quinone) cm-1. Crisamicin C plasma desorption mass spectrometry (PD-MS) m/z 615.9 [M + H)+, hydroquinone) was 16 amu higher than crisamicin A PD-MS m/z 600 [M + H)+, hydroquinone) suggesting that the two antibiotics differ by one additional oxygen in crisamicin C. Analysis of 1H and 13C NMR spectra, in comparison with those of crisamicin A, indicated that crisamicin C was the 4'a, 10'a epoxide derivative of crisamicin A. Carbon-thirteen labeled acetate feeding experiments were used to confirm the positions of the epoxide and other structural features. Crisamicin C was a more potent antibiotic than crisamicin A, but shared the same spectrum of antimicrobial activity (Gram-positive only).

Production of nanaomycin and other antibiotics by phosphate-depressed fermentation using phosphate-trapping agents.

Nanaomycin production by Streptomyces rosa subsp. notoensis in complex media was inhibited by exogenously supplied inorganic phosphate. The inhibition was reversed by phosphate-trapping agents such as allophane and aluminum oxide. Under such condition nanaomycin production increased to the control level, and the phosphate content dropped down to the unsupplemented level. When allophane was added to conventional complex media containing nutrient-derived inorganic phosphate, the production of nanaomycin and several other antibiotics, which are subject to phosphate regulation, was enhanced several fold with the simultaneous reduction of free phosphate. The term "phosphate-depressed fermentation" is proposed for this technique.

New anticoccidial antibiotics, WS-5995 A and B. I. Isolation and characterization.

WS-5995 A, B and C are produced by a new strain of Streptomyces designated Streptomyces auranticolor. These antibiotics were purified by solvent extraction followed by chromatography on silica gel and then crystallized. WS-5995 A (C19H12O6, m.p., 289 approximately 291 degrees C) and WS-5995 B (C19H14O6, sublimation at 300 degrees C) protect chickens from infection with Eimeria tenella, a species of coccidia, which produces morbidity or mortality in chickens. WS-5995 C (C19H14O 7, m.p. 288 approximately 290 degrees C), a biologically inactive component, was found to be converted to WS-5995 A on treatment with trifluoroacetic anhydride.

Isolation and characterization of sarubicin A, a new antibiotic.

The new antibiotic sarubicin A [red crystals, mp. 194 approximately 195 degrees C, C18H14N2O6 (I)] was isolated from fermentations of a Streptomyces strain. The compound is moderately active in vitro against Micrococcus luteus.

Gunacin, a new quinone antibiotic from Ustilago sp.

In a screening program for antibiotics which were antagonized by cysteine, a strain, which was characterized as Ustilago sp., was found to produce a new quinone antibiotic, gunacin. The molecular weight M+ = 348.084 determined by mass spectroscopy, corresponds to a molecular formula of C17H16O8. Further spectroscopic data prove that gunacin is a new antibiotic. The antibiotic possesses a good inhibitory effect against mycoplasmas and Gram-positive bacteria including multi-resistant strains. It also possesses a weak activity against Gram-negative bacteria with the exception of Proteus vulgaris, which is more strongly inhibited. The main activity against fungi is found against Trichophyton mentagrophytes. Gunacin shows an inhibition of the DNA synthesis in vivo, is antagonized by mercapto compounds and possesses an acute toxicity of LD50 = 16 mg/kg i.p. and LD50 = 12 mg/kg i.v. in mice. Against HeLa-cell the antibiotic shows an ED50 = 12.11 microgram/ml. Thirty five microgram/ml of gunacin induces 1,063 interferon units.

2,2'-dimethoxy-4a,4a'-dehydrorugulosin (rugulin), a minor metabolite from Penicillium rugulosum.

A new metabolite was isolated from the culture of Penicillium rugulosum and its structure was determined from physico-chemical data. Accompanying metabolites skyrin and rugulosin were characterized by UV, IR, CD, mass and NMR spectra.