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 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).

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.

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.

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.

Homology between Streptomyces genes coding for synthesis of different polyketides used to clone antibiotic biosynthetic genes.

Many important antibiotics such as tetracyclines, erythromycin, adriamycin, monensin, rifamycin and avermectins are polyketides. In their biosynthesis, multifunctional synthases catalyse iterated condensation of thio-esters derived from acetate, propionate or butyrate to yield aliphatic chains of varying length and carrying different alkyl substituents. Subsequent modifications, including aromatic or macrolide ring closure or specific methylations or glycosylations, generate further chemical diversity. It has been suggested that, if different polyketide synthases had a common evolutionary origin, cloned DNA coding for one synthase might be used as a hybridization probe for the isolation of others. We show here that this is indeed possible. Study of a range of such synthase genes and their products should help to elucidate what determines the choice and order of condensation of different residues in polyketide assembly, and might yield, by in vitro recombination or mutagenesis, synthase genes capable of producing novel antibiotics. Moreover, because genes for entire antibiotic pathways are usually clustered in Streptomyces, cloned polyketide synthase genes are valuable in giving access to groups of linked biosynthetic genes.

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.

Production of new hybrid antibiotics, mederrhodins A and B, by a genetically engineered strain.

Hybrid antibiotics mederrhodins A and B were produced by a recombinant strain consisting of the medermycin-producing Streptomyces sp. strain AM7161 containing part of the gene clusters for actinorhodin biosynthesis of Streptomyces coelicolor A3(2). Mederrhodin A has a hydroxyl group at the C-6 position of the medermycin molecule, and mederrhodin B is dihydromederrhodin A. The antimicrobial activity of mederrhodin A resembled that of medermycin. Mederrhodin B was almost devoid of antimicrobial activity.

Further studies on the biosynthesis of granaticin.

Experiments with cerulenin-inhibited cultures of S. violaceoruber showed conversion of dihydrogranaticin (II) into granaticin (I), but not vice versa, confirming an earlier conclusion that II is the biosynthetic precursor of I. Feeding of CH3(13)C18O2Na followed by 13C-NMR analysis of the product by the 18O shift method indicated the expected incorporation of 18O at carbons 1, 11 and 13 of I and showed that the oxygen of the pyran ring originates from C-3 and not from C-15. Analysis of I biosynthesized from 13C2H3COONa by 13C[1H, 2H] triple resonance NMR spectroscopy showed the incorporation of one atom of deuterium each at C-2 and C-4. C-16 carried a maximum of 2, not 3, atoms of deuterium. These results are discussed in terms of biosynthetic mechanisms.

Conversion of 1-naphthol to naphthoquinone metabolites by rat liver microsomes: demonstration by high-performance liquid chromatography with reductive electrochemical detection.

1-Naphthol has recently been shown to be selectively toxic to short-term organ cultures of human colorectal tumor tissue. The mechanism underlying 1-naphthol's selective toxicity is as yet unknown, but may be due to the formation of naphthoquinone metabolites, which are known to be highly toxic to tumor cells. By using high-performance liquid chromatography with reductive electrochemical detection, it has been possible to show that 1-naphthol is converted to naphthoquinone metabolites by rat liver microsomes. At least two metabolic pathways, independent of cytochrome P-450, appear to be involved. Iron-dependent lipid peroxidation appears to be responsible for at least part of the conversion of 1-naphthol to predominantly 1,4-naphthoquinone, and it seems likely that superoxide anion radical generation by NADPH-cytochrome P-450 reductase could also catalyze this conversion. 1-Naphthol therefore seems to be converted to cytotoxic naphthoquinone metabolites by mechanism(s) dependent upon the generation of free radicals in rat liver microsomes. The results also demonstrate the utility of HPLC with reductive electrochemical detection for investigations of quinone metabolite formation and the measurement of quinones of both physiological and environmental interest.

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.

A selective and indicative medium for groups of Penicillium viridicatum producing different mycotoxins in cereals.

A medium, pentachloronitrobenzene-rose bengal-yeast extract-sucrose agar (PRYES), for the isolation of moulds occurring during storage of cereals has been developed and compared with other selective media. The basal medium is yeast extract agar containing 15% sucrose (w/v). In addition to the sucrose content further selective measures include the addition of antibacterial antibiotics chloramphenicol and chlortetracycline (50 mg/l), the fungicides rose bengal (25 mg/l each), and pentachloronitrobenzene (1 g/l) and a low incubation temperature (20 degrees C). Members of the Mucorales were completely inhibited, and fast-growing species of other moulds were slightly inhibited, allowing important storage moulds to develop. The important ochratoxin A and citrinin-producing Penicillium viridicatum group II was indicated by a typical violet brown reverse on PRYES. Producers of xanthomegnin and viomellein (P. viridicatum group I and P. aurantiogriseum) were indicated on PRYES by their yellow reverse and obverse colours. The medium was used for screening 40 samples of barley, and moulds with the characteristic colours were all identified as the species mentioned above.

Isolation of xanthomegnin from Penicillium viridicatum by preparative high-pressure liquid chromatography.

A method was developed for the production and purification of xanthomegnin from Penicillium viridicatum (NRRL 6430) cultured on rice at 15 degrees C for 29 days. Liquid-liquid extraction followed by high-pressure liquid chromatography afforded 440 mg of crystalline xanthomegnin per kg of rice.

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.

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.