Penitricin, a new class of antibiotic produced by Penicillium aculeatum. IV. Biosynthesis.

Bioconversion experiments using washed mycelia of Penicillium aculeatum NR 5165 and NR 6216 revealed that penitricin (Ro 09-0804, hydroxymethylcyclopropenone) was biosynthesized from trans-2-butene-1,4-diol via 4-hydroxycrotonaldehyde.

Studies on the biosynthesis of basic 16-membered macrolide antibiotic, platenomycins. I. Selection of and cosynthesis by non-platenomycin-producing mutants.

In a search for blocked mutants which may produce a biosynthetic intermediate, mutation by N-methyl-N'-nitro-N-nitrosoguanidine treatment and/or ultra-violet irradiation were performed on platenomycin-producing Streptomyces platensis subsp. malvinus MCRL 0388 (NRRL 3761). Twenty four non-platenomycin-producing stable mutants were thus obtained and tested for cosynthesis ability. Antibiotic cosynthesis with a pair of these mutants made it possible to detect the producer of an intermediate. Among these mutants which were classified into eight groups (A to G and doubtful groups), mutants of groups A and B appeared from their complementation pattern to be the useful producers of biosynthetic intermediates of platenomycin.

Studies on the biosynthesis of basic 16-membered macrolide antibiotics, platenomycins. II. Production isolation and structures of 3-O-propionyl-5-O-mycaminosyl platenolides I and II, 9-dehydro demycarosyl platenomycin and demycarosyl platenomycin.

Four basic glycosides have been isolated from the fermentation broth of the blocked mutants of Streptomyces platensis subsp. malvinus MCRL 0388. These compounds isolated and purified by solvent extraction and column chromatography were identified as 3-O-propionyl-5-O-mycaminosyl platenolides I (PPL-I-MC) and II (PP-II-MC), 9-dehydro demycarosyl platenomycin (DDM-PLM) and demycarosyl platenomycin (DM-PLM).

Studies on the biosynthesis of basic 16-membered macrolide antibiotics, platenomycins. III. Production, isolation and structures of platenolides I and II.

Two neutral macrocyclic lactones designated platenolides I and II have been isolated as the major products from the fermentation broth of the blocked mutants of Streptomyces platensis subsp. malvinus. These two compounds were isolated by solvent extraction and purified by column chromatography. Both platenolides [PL-I: C20H32O6, PL-II: C20H34O6] are closely related to the platenomycin aglycone.

Studies on the biosynthesis of basic 16-membered macrolide antibiotics, platenomycins. IV. Biosynthesis of platenomycins.

To elucidate the biosynthetic pathway of platenomycin (PLM), biosynthetic relationships of platenolides I (PL-I) and II (PL-II), 3-O-propionyl-5-O-mycaminosyl platenolides I (PPL-I-MC) and II (PPL-UU-MC), 9-dehydro demycarosyl platenomycin (DDM-PLM) were examined with growing cultures or the washed mycelium of blocked mutants of Streptomyces platensis subsp. malvinus MCRL0388, a platenomycin-producing organism. As a result, it was revealed that PLM was biosynthesized from PL-I via DM-PLM and DA-PLM along the pathways shown in Chart 1. 4''-Isovaleroyl unit of PLM-A and 4''-propionyl unit of PLM-B were respectively derived from L-leucine and L-isoleucine.

Function of plasmids in the production of aureothricin. I. Elimination of plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis.

The spontaneous mutant 18a derived from Streptomyces kasugaensis MB273 exhibited pleiotropic effect such as loss of aerial mycelium formation, aureothricin (AT) production, and of citrullin biosynthesis, as well as changes in plasmid; the mutant required cystine for production of aureothricin. An improved method of protoplast regeneration was applied to S. kasugaensis MB 273-18a and a regeneration efficiency of 90% or more was obtained. Sixty to ninety percent of the colonies regenerated from the 18a protoplasts exhibited reversion of the pleiotropic mutation in 18a. Moreover, of 13 regenerated strains which showed these drastic phenotypic variations, it was found that their plasmid types varied. These types could be divided into two groups; the RI type (5 strains) which contained a large amount of pSK2, a small amount of pSK3 and no pSK1, and the RII type (8 strains) in which no closed-circular DNA was detected. From these results, the following conclusions were obtained. First, plasmid curing in RII type strains and also the variation of plasmid copy in the RI type strains occurred as the result of protoplast regeneration. Second, the structural genes for biosynthesis of AT probably exist on chromosome. Third, regeneration of 18a protoplasts causes the reversion of pleiotropic mutation with high frequency. A working hypothesis was proposed to explain these complex phenomena.

Penitricin, a new class of antibiotic produced by Penicillium aculeatum. III. Structural confirmation by chemical synthesis and biological activity.

A novel antibiotic, penitricin showing anti-Gram-negative activity, has been isolated from the culture filtrate of Penicillium aculeatum. Chemical and physico-chemical studies determined the structure of penitricin as hydroxymethylcyclopropenone (1), which was confirmed by chemical synthesis from propargyl alcohol. Biological activity of penitricin and several cyclopropenones as well as two metabolites, penitricins B and C was compared.

TPU-0031-A and B, new antibiotics of the novobiocin group produced by Streptomyces sp. TP-A0556.

Two novel antibiotics, TPU-0031-A and B, were isolated from the culture broth of an actinomycete strain. The producing strain, TP-A0556, was identified as Streptomyces sp. based on the taxonomic study. The new antibiotics were obtained by solvent extraction and chromatographic purification. Spectroscopic analyses showed that TPU-0031-A and B were 7'-demethylnovobiocin and 5"-demethylnovobiocin, respectively. These compounds showed antibiotic activity against Gram-positive and -negative bacteria.

Cedarmycins A and B, new antimicrobial antibiotics from Streptomyces sp. TP-A0456.

Two novel butyrolactone antibiotics, cedarmycins A and B, were isolated from the cultured broth of the actinomycete Streptomyces sp. TP-A0456. The new compounds were purified by HP-20 resin, silica gel, ODS column chromatographies and preparative HPLC, consecutively. The structure of cedarmycin was determined by spectroscopic methods as an alpha,beta-unsaturated butyrolactone with a fatty acid side chain. These compounds showed antibiotic activity against Gram-positive and -negative bacteria and yeasts. Among the tested organisms, cedarmycins potently inhibited the growth of Candida glabrata IFO 0622 with the MIC of 0.4 microg/ml, comparable to that of amphotericin B.

Mass spectrometry of platenolides and their derivatives in connection with structure elucidation.

Mass spectra of platenolides--biosynthetic precursors of the 16-membered macrolide antibiotics, platenomycins--and their derivatives are discussed in detail especially in connection with structure elucidation. Mass spectrometry was of great use in establishing the structures for platenolides I (1) and II (2).

Mutational biosynthesis of butirosin analogs. I. Conversion of neamine analogs into butirosin analogs by mutants of Bacillus circulans.

By N-methyl-N'-nitro-N-nitrosoguanidine treatment, neamine-negative mutants which required neamine for biosynthesis of butirosins were obtained from a butirosin-producing organism Bacillus circulans. These mutants also produced butirosins from paromamine and could be divided into two types I and II. Mutants of type I could not produce butirosins from 2-deoxystreptamine, whereas those of type II could. Two typical mutants MCRL 5003 (type I) and MCRL 5004 (type II) could produce butirosin analogs, 3', 4'-dideoxybutirosins, 6'-N-methylbutirosins, 3', 4'-dideoxy-6'-N-methylbutirosins and 3', 4'-dideoxy-6'-C-methyl-butirosins from neamine analogs, gentamine Cla, 6'-N-methylneamine, 6'-N-methylgentamine Cla and gentamine C2, respectively.

Penitricin, a new class of antibiotic produced by Penicillium aculeatum. I. Taxonomy of the producer strain and fermentation.

A novel antibiotic, penitricin (Ro 09-0804) was discovered in the culture filtrate of a fungal strain NR 5165. Taxonomic studies of the producing organism resulted in its assignment to Penicillium aculeatum. Further examination on penitricin production by other strains of this species and related taxa revealed that penitricin was produced by several other strains of P. aculeatum, but not by any available strains of the closely similar species, P. verruculosum. It was also found that copper ion was essential for production of penitricin.

Eurystatins A and B, new prolyl endopeptidase inhibitors. III. Fermentation and controlled biosynthesis of eurystatin analogs by Streptomyces eurythermus.

Accurate and precise component analysis of eurystatin analogs in fermentation broth was devised by HPLC methods with and without 2,4-dinitrophenylhydrazonation. Detailed optimization of fermentation conditions and strain improvement by HPLC analysis significantly increased the eurystatin productivity of Streptomyces eurythermus. Chemically defined fermentation media which produced eurystatins A and B at fermentation yields comparable to complex media were elaborated for radio-isotope fermentation studies and controlled biosynthesis. Radio-isotope incorporation study using 14C-labeled amino acids in chemically defined medium demonstrated that L-leucine and L-ornithine were the direct precursors for the L-leucine and L-ornithine moieties of eurystatins A and B, respectively. Based on this finding, L-valine and L-isoleucine were supplemented to the growing culture of S. eurythermus in chemically defined medium, which resulted in the controlled biosynthesis of new eurystatin analogs named eurystatins C, D, E and F.

Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streptomycetes. I. Purification and characterization.

Streptomycetes, which belong to the Gram-positive bacteria, produce secondary metabolites and sporulate. The timing of starting the secondary metabolite production and the sporulation depends on environmental conditions such as nitrogen and carbon sources. In order to obtain a tool for understanding the regulation mechanism, we carried out screening for chemical substances that induce secondary metabolism and sporulation in streptomycetes and found an active substance from the culture broth of Streptomyces sp. TP-A0584. This substance designated goadsporin promoted the formation of red pigment and sporulation at a concentration of 1 microM in Streptomyces lividans TK23 which does not produce the pigment under normal growth conditions. Goadsporin is an oligopeptide consisting of 19 amino acids with the molecular formula C72H97N19020S2. Sporulation and/or secondary metabolite production was induced in 36 streptomycetes strains among 42 strains tested. These results suggest that goadsporin acts on a common regulation pathway for sporulation and secondary metabolism in streptomycetes and can be a powerful tool to analyze the regulation mechanism.

Arisostatins A and B, new members of tetrocarcin class of antibiotics from Micromonospora sp. TP-A0316. I. Taxonomy, fermentation, isolation and biological properties.

Arisostatins A and B, new members of tetrocarcin class of antibiotics were isolated from the culture broth of an actinomycete strain. The producing strain, TP-A0316, was identified as Micromonospora sp. Arisostatins were obtained from the culture fluid by solvent extraction and chromatographic purification. They showed antibiotic activity against Gram-positive bacteria and antitumor activity.

High-mannose type oligosaccharide-dependent apoptosis in U937 cells induced by pradimicin, a mannose-binding antibiotic.

Cell surface oligosaccharides play a role in a variety of biological events such as cell adhesion and signal transduction. We have shown that BMY-28864, a semi-synthetic analog of pradimicin, induced apoptosis of U937 cells which had been incubated with 1-deoxymannojirimycin, an inhibitor of mannosidase I. BMY-28864 was not cytotoxic to the cells which had been cultivated with other glycosidase inhibitors such as castanospermine and swainsonine. We thus propose that BMY-28864 induces apoptosis by acting on a specific mannose-rich oligosaccharide, presumably (Man)9(GlcNAc)2+.

Involvement of Ca2+ ion and reactive oxygen species as a mediator in pradimicin-induced apoptosis.

Pradimicin (PRM) induces apoptosis in mammalian cells which had been incubated with 1-deoxymannojirimycin (DMJ). Flow cytometric analysis revealed that PRM preferentially induced apoptosis to the cells of the G1 phase. Two possible mediators in this apoptotic cascade were identified. Exposure of DMJ-treated cells to PRM resulted in a rapid (approximately 5 seconds) and slow (approximately 30 minutes) elevation of the intracellular calcium level. Reactive oxygen species (ROS) were proved to be involved in this system by the fact that the apoptosis was completely inhibited by treating the cells with a ROS scavenger, N-acetylcysteine in prior to the PRM stimulation.

Macrolide antibiotics M-4365 produced by Micromonospora. I. Taxonomy, production, isolation, characterization and properties.

A series of basic 16-membered macrolide antibiotics, M-4365 A1, A2, A3, G1, G2 and G3, were isolated from the culture broth of strain MCRL 0940 which is assigned to be a new species of Micromonospora and for which the name Micromonospora capillata sp. nov. is proposed. Among these antibiotics, M-4365 A2 and G2 showed strong inhibitory activity against Gram-positive and Gram-negative bacteria.

DNA homology studies in Streptomyces using S1 nuclease.

The optimal reaction conditions for the determination of DNA-homology in Streptomyces species were established in the presence of formamide using S1 nuclease. The melting temperature of Streptomyces DNA was 90 degrees C in 0.42 M NaCl containing 20% formamide in which the denaturation was completed by boiling for 5 minutes. In the S1 reaction mixture consisting of 5 U of S1 nuclease, 0.168 M NaCl, 1 mM ZnSO4 and 8% formamide at pH 4.8, single-stranded DNA was hydrolyzed by more than 98%, while the hydrolysis of double-stranded DNA was less than 3%. From the analysis of homoduplex formation, the C0t1/2 was found at 20 hours, when a mixture of unlabeled DNA and index DNA was used at a ratio of 500:1.

Penitricin, a new class of antibiotic produced by Penicillium aculeatum. II. Isolation and characterization.

A novel antibiotic, penitricin, Ro 09-0804, has been produced in the culture filtrate of Penicillium aculeatum NR 5165. This antibiotic was purified by repeated extraction of culture filtrate with 1-butanol, and passage of the crude extracts through Sephadex G-10, followed by HPLC (Shodex Ionpak S-801). Physico-chemical characterization was made on penitricin, while two open-ringed penitricins B and C co-produced by the producer strain were also identified.