Studies on new phosphate ester antifungal antibiotics phoslactomycins. I. Taxonomy, fermentation, purification and biological activities.

New antibiotics phoslactomycins A, B, C, D, E and F, which contain alpha, beta-unsaturated delta-lactone, phosphate ester, conjugated diene and cyclohexane ring moieties, were isolated from the culture broth of a soil isolate actinomycete. Morphological, cultural and physiological studies revealed that the isolate is a strain of Streptomyces nigrescens. Phoslactomycins were obtained by butanol extraction, gel filtration and reverse phase chromatography. The antibiotics show strong activity against various fungi, particularly phytopathogenic fungi (Botrytis cinerea and Alternaria kikuchiana).

Studies on new phosphate ester antifungal antibiotics phoslactomycins. II. Structure elucidation of phoslactomycins A to F.

Phoslactomycins A to F are new antifungal antibiotics produced by Streptomyces nigrescens SC-273. On the basis of physico-chemical properties and NMR studies, their structures have been determined as shown in Fig. 6. They are characterized by possessing alpha, beta-unsaturated delta-lactone, phosphate ester, conjugated diene and cyclohexane ring moieties. The structural difference between them is ascribed to a substituent bound to the cyclohexane ring.

Oxidases involved in biosynthesis of macrolide antibiotic patulolides from Penicillium urticae S11R59.

Patulolides are a group of 12-membered macrolide antibiotics produced by Penicillium urticae S11R59. An enzyme involved in the conversion of patulolide C to patulolide A was purified from P. urticae S11R59 and characterized. The enzyme showed a single band on SDS-PAGE and molecular sieve HPLC both of which indicated a Mr of 86,000, indicating that the enzyme is monomeric. However, the enzyme was separated into two bands of very similar pI's (pI 4.2 and 4.3) by isoelectric focusing. Both bands catalyzed the conversion of patulolide C to patulolide A, as demonstrated by activity staining. The two isoenzymes were proved to be oxidases by the simultaneous production of H2O2 during the conversion of patulolide C to patulolide A. The molar ratio for patulolides C, A and H2O2 was determined to be 1:1:1. The optimum pH and temperature were determined to be 7 and 35-40 degrees C, respectively, and the enzymes were stable at pH 6-9 and 4-40 degrees C. The oxidases showed characteristic absorption at 345 and 450 nm, indicating the presence of flavin as coenzyme. Among several analogues of patulolide C tested, the oxidases showed very narrow substrate-specificity; only patulolide C was oxidized to patulolide A. No enzyme activity for the reverse reaction, i.e. from patulolide A to patulolide C, was present in the cell-free extract of P. urticae S11R59. Patulolide C oxidases therefore play a key role in the biosynthesis of patulolides.

Biosynthetic origin of the carbon skeleton and oxygen atoms of the LL-F28249 alpha, a potent antiparasitic macrolide.

The biosynthesis of LL-F28249 alpha in a culture of Streptomyces cyaneogriseus has been studied using 13C, 14C and 18O labeled precursors. A complete 13C NMR spectrum of F28249 alpha has been assigned. Incorporation studies using 13C labeled precursors indicate that the carbon skeleton of F28249 alpha is derived from seven acetate, six propionate and one 2-methylpropionate units. The origin of the oxygen atoms of F28249 alpha has been examined by feeding [1-13C,18O2]acetate, [1-13C,18O2]propionate, [2-13C]acetate/18O2 and 18O2 separately to the fermentation culture and analyzing the resulting labeled LL-F28249 alpha samples by 13C NMR, electron impact MS and chemical ionization MS. Out of a total of eight oxygen atoms in LL-F28249 alpha, four oxygen atoms are derived from acetate, three from propionate and one from molecular oxygen.

Biosynthesis of the macrolide antibiotic patulolides by Penicillium urticae S11R59: identification of the origin of carbon atoms by 13C NMR spectroscopy.

Patulolides are 12-membered macrolides produced by Penicillium urticae S11R59, and they are the simplest macrolide antibiotics. All carbon signals, including six methylene signals of patulolides A (1), B (2), and C (3), were completely assigned by use of the 13C two-dimensional INADEQUATE. The biosynthesis of patulolides was investigated with 13C labeled acetate. Feeding of [1-13C]acetate to a culture of P. urticae S11R59 gave patulolides A (1), B (2), C (3), each of which showed enrichment at carbons 1, 3, 5, 7, 9, and 11; enrichment at carbons 2, 4, 6, 8, 10, and 12 was observed upon feeding of [2-13C]acetate. These results showed that patulolides A (1), B (2), and C (3) are pure acetogenic hexaketides derived from six acetate units coupled in head-to-tail fashion.

Nitrogen inhibition of mycotoxin production by Alternaria alternata.

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.

Novel macrocyclic antibiotics: megovalicins A, B, C, D, G and H. I. Screening of antibiotics-producing myxobacteria and production of megovalicins.

New antibiotics belonging to macrocyclic were discovered and named megovalicins A, B, C, D, G and H. The antibiotics were accumulated endogeneously by a newly isolated myxobacterium identified as Myxococcus flavescens. Tank culture of the bacterium for 4 days gave 8.8 g cells/liter on a wet basis, and 4.8, 7.1, 20.0, 0.4, 3.75 and 15.0 micrograms of megovalicins A, B, C, D, G and H respectively were obtained from 1 g wet cells.

Lipstatin, an inhibitor of pancreatic lipase, produced by Streptomyces toxytricini. I. Producing organism, fermentation, isolation and biological activity.

Lipstatin, a new and very potent inhibitor of pancreatic lipase (the key enzyme of intestinal fat digestion) was isolated from Streptomyces toxytricini. Lipstatin contains a beta-lactone structure that probably accounts for the irreversible lipase inhibition. The IC50 of lipstatin for pancreatic lipase is 0.14 microM. In mice triolein absorption was dose-dependently inhibited by lipstatin, whereas oleic acid was absorbed normally. Other pancreatic enzymes, such as phospholipase A2 and trypsin, were not inhibited even at an inhibitor concentration of 200 microM.

Phomenoic acid and phomenolactone, antifungal substances from Phoma lingam (Tode) Desm.: kinetics of their biosynthesis, with an optimization of the isolation procedures.

Phomenoic acid and phomenolactone, previously isolated from the fungus Phoma lingam (Tode) Desm., have shown moderate antifungal and antibacterial properties in vitro. To rationalize the production of phomenoic acid, a kinetic study of its biosynthesis in the mycelium was performed. Phomenoic acid and phomenolactone appear in the mycelium after a prolonged incubation, a phenomenon which may be of particular interest for the production of these substances or in the study of the mechanism of their biosynthesis. The isolation procedure was optimized for phomenoic acid. Through a series of SiO2 column chromatographies, high-pressure liquid chromatography, and transformation of the mixture of phomenoic acid and phomenolactone into methyl phomenoate (BF3), the final yield of phomenoic acid reached 160 mg/liter of culture medium. An alternative method for the isolation of both phomenoic acid and phomenolactone is also reported in detail.

Biosynthesis of the avermectins by Streptomyces avermitilis. Incorporation of labeled precursors.

The biosynthesis of the avermectins, a group of 16 membered macrolides with potent anthelmintic and insecticidal activity produced by Streptomyces avermitilis, was studied by supplying cultures with 14C and 13C precursors. [1-14C] and [2-14C]acetate and propionate were poor precursors of the avermectins and were instead rapidly oxidized to 14CO2. The S-methyl of methionine in contrast was incorporated extensively and equally into the three methoxyl groups of the avermectins. The carbon backbone of methionine was not a precursor of the avermectins. Feeding of [1-13C]glucose yielded avermectins labeled specifically in the C1' and C1" of the oleandrose moiety and in the aglycone moiety in carbons known to be derived from the methyl of acetate. Feeding [U-13C]glucose showed that the entire avermectin molecule is derived from glucose carbons.

Demethylavermectins. Biosynthesis, isolation and characterization.

Streptomyces avermitilis normally produces eight avermectins. Avermectin A components contain three methoxyl groups; two on the oleandrose disaccharide and one on the aglycone moiety at C5. Avermectin B components contain methoxyl groups only on the oleandrose disaccharide. Sinefungin inhibits methylation at all three sites. Addition of sinefungin to S. avermitilis Agly-1, a mutant which produces virtually only avermectin aglycone A components, alters the fermentation and causes an accumulation of avermectin aglycone B components. Addition of sinefungin to S. avermitilis 08, a high producing strain, results in accumulation of 8 new avermectins which lack methoxyl groups on the oleandrose moieties as well as the aglycone. These new avermectins were isolated and shown to possess anthelmintic and insecticidal activity.

Isolation and preliminary characterization of lytic and lysogenic phages with wide host range within the streptomycetes.

Examination of approximately 700 soil samples yielded about 100 actinophages. Restriction analysis of phage DNA indicated that 57 are unique, and of these, 20 produce turbid plaques on one or more of the streptomycetes tested. Five phages are shown to insert into the genome of Streptomyces avermitilis. None of the phages was able to perform generalized transduction of S. avermitilis.

Biosynthesis of the antibiotic maduramicin. Origin of the carbon and oxygen atoms as well as the 13C NMR assignments.

The biosynthesis of maduramicin alpha and beta in a culture of Actinomadura yumaensis has been studied using 13C, 14C and 18O labeled precursors. The alpha component of this recently discovered polyether antibiotic, containing forty-seven carbon atoms in a seven-ring system, is derived from eight acetate, seven propionate and four methionine molecules. The beta component which is missing one methoxy group incorporates three methionine methyl groups. The carbohydrate moiety was enriched by methionine, but not significantly by acetate or propionate. Studies of the incorporation of 13C labeled precursors permit the 13C NMR assignment of maduramicin. The origin of oxygen atoms of maduramicin has been examined by feeding [1-13C, 18O2]acetate and [1-13C, 18O2]propionate separately in the fermentation culture and the resulting doubly labeled maduramicin samples were analyzed by the isotopic shifts in the 13C NMR spectra. These results are consistent with the initial formation of a triene, which is converted to maduramicin by cyclization of the triepoxide.

Studies on new antifungal antibiotics, guanidylfungins A and B. I. Taxonomy, fermentation, isolation and characterization.

Guanidylfungin A, C58H103N3O18, and guanidylfungin B, C57H101N3O18, were isolated from the mycelia of Streptomyces hygroscopicus No. 662 by means of silica gel absorption and reversed phase liquid chromatographies. The guanidylfungins were active against fungi and Gram-negative bacteria.

Studies on new antifungal antibiotics, guanidylfungins A and B. II. Structure elucidation and biosynthesis.

The structures of guanidylfungins A and B were elucidated from the physico-chemical properties of these compounds and the structures of the degradation products by ozonolysis and periodate oxidation. The guanidylfungins consist of a 36-membered polyhydroxyl lactone ring, a guanidine and a monoester of malonic acid. The labelling experiments with sodium [1-13C]acetate and sodium [1-13C]propionate revealed that twelve units of acetate and nine of propionate were incorporated into the molecule of guanidylfungin A.