A genetic recombination study of an industrial strain of Streptomyces avermitilis which produces avermectin is described. A genetic map has been constructed by analysis of haploid recombinants and linkage relationships of 16 marker loci. Fifteen avermectin-nonproducing mutants, produced by mutagenesis, were classified into two phenotypically different groups, of which one produced avermectin aglycon and the other was able to convert avermectin aglycon to avermectins. Two different mutants were found to map closely to each other.
The addition of glucose in the early stage of fermentation suppressed not only avermectin production but also the activity of 6-phosphogluconate dehydrogenase in the pentose phosphate pathway. On the other hand, when glucose was added at the late stage of fermentation, suppression of avermectin formation and 6-phosphogluconate dehydrogenase activity was not observed but avermectin formation was increased and about a twofold-higher content of avermectins than that of the control fermentation was accumulated.Antibiotic production, in general, is regulated by various parameters, e.g., medium, aeration, and temperature, and the production of some antibiotics is suppressed by glucose, ammonium ions, and phosphate ions (6).In the present paper, we describe the effect of glucose on avermectin (1, 3, 4, 7) production by Streptomyces avermitilis and on the production of some enzymes related to sugar metabolism.S. avermitilis K139 and its derivatives K475 and K655, which are high-yield mutants, were used in this experiment. The spores of the strains, which grew on YMS medium containing 4 g of yeast extract (Difco Laboratories), 10 g of malt extract (Difco), 4 g of soluble starch (Difco), and 20 g of Bacto-Agar (Difco) in 1 liter of distilled water (pH 7.4), were scraped and stored in 20% (wt/vol) glycerol at -30°C. A portion of the spores was inoculated into a 50-ml test tube containing 10 ml of seed medium (3) and a stainless spring coil and cultured at 28°C for 48 h on a reciprocal shaker to give a seed culture. Then 0.3 ml of the seed culture was inoculated into a 100-ml Erlenmeyer flask containing 10 ml of production medium (3) and cultured at 28°C on a rotary shaker. The content of glucose in the culture was determined by an enzymatic method using glucose oxidase and peroxidase. After termination of the fermentation, an equal volume of methanol was added to the culture and the mixture was shaken for 30 min to extract avermectins from the mycelium. A 5-,ul sample of the supernatant of methanol extract was examined by high-performance liquid chromatography. A column (6.54) by 75 mm [inner diameter]) was packed with ODS-Hypersil-3 (3 ,um) and developed with methanol-water (85:15 [vol/vol]) at a flow rate of 0.9 ml/min in ambient conditions. The quantities of avermectins were calculated from the integration value at 246 nm using an authentic sample of avermectin Bla as a standard. The crude enzyme preparation was prepared as described by Ikeda et al. (5). The assay for glucose kinase was done as described by Ikeda et al. (5). Phosphofructokinase was assayed by the method described by Seno and Chater (9), except that fructose-6-phosphate was used as substrate. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were assayed as described by Stowers and Elkan (10). The assay of citrate synthase was done as described by Ochoa et al. (8). The protein concentration of the enzyme preparation was determined by the method of Bradford (2).As shown in Fig. 1, the rate of glucose consumption in the * Corresponding...
A new series of antifungal antibiotics, Ro 09-1470 and its 6 congeners were isolated from the fermentation broth of Penicillium sp. NR6564. Their structures were determined as tetrahydropyran derivatives with an alkenyl side chain on the basis of their spectroscopic and physico-chemical properties. Among these compounds, Ro 09-1470 and Ro 09-1545 possessing a glycyl or an TV-substituted glycyl ester residue had high antifungal activity. Ro 09-1469, one of the congeners, was found in the fermentation broth of several strains of Aspergillus sclerotiorum Huber.
A novel cholecystokinin type-B receptor antagonist named tetronothiodin has been isolated by column chromatography and preparative HPLCfrom the fermentation broth of Streptomyces sp. NR0489. Tetronothiodin inhibited the binding of CCK8(C-terminal octapeptide of cholecystokinin) to rat cerebral cortex membranes (CCK type-B receptors) with an IC50 of 3.6nM, whereas it did not inhibit CCK8binding to rat pancreatic membranes (CCK type-A receptors). It also inhibited CCK8induced Ca2+ mobilization in GH3cells, a rat anterior pituitary cell line, but was without effect on the basal cytosolic Ca2+ concentration. This finding indicated tetronothiodin was an antagonist of CCKtype-B receptors. Cholecystokinin(CCK) is a hormonal regulator of pancreatic secretion1* as well as gallbladder contraction2* and gut motility3). It has also been proposed to act as a neurotransmitter in the central nervous system4*. CCKtype-B (CCK-B)receptors are suggested to be related to appetite5*, pain6'7* and anxiety8'9*. SomeCCK-Breceptor antagonists increased food intake5*, enhanced morphine analgesia6'7* and reduced anxiety8'9* in rats. However, physiological and pharmacological roles of CCK-Breceptorsare not yet fully understood in part because of the shortage of potent and specific CCK-B receptor antagonists. To obtain structurallv unique and specific CCK-Breceptor antagonists, we screened microbial metabolites by employing a binding assay method in which rat cerebral cortex membranesand 125I labeled Bolton-Hunter CCK8 ([125I]-CCK8) were used as the receptors and the radioligand, respectively. In this screening program, we discovered a novel CCK-B receptor antagonist named tetronothiodin (1) from the culture broth of Streptomyces sp. NR0489, and determined the structure to be a macrocyclic compound containing an aacyltetronic acid and a tetrahydrothiophene ring (Fig. 1). A preliminary communication of this work Fie. 1. Structure of tetronothiodin (1
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