Lloyd E. McDaniel

Factors Affecting the Production of Candicidin

Factors affecting candicidin synthesis and mycelial growth of Streptomyces griseus IMRU 3570 were studied. Inorganic phosphate was found to inhibit candicidin synthesis but to stimulate mycelial growth. Zinc, iron, and magnesium ions stimulated candicidin synthesis at relatively high concentrations in a complex medium but not in a synthetic medium. No other factors studied, such as temperature, oxygen absorption rate, and sugar concentration, were found to differentially affect antibiotic synthesis and mycelial growth. Optimum concentration of inorganic phosphate for candicidin synthesis in a chemically defined medium was found to be between 5 × 10−5 and 5 × 10−4 M. The culture in idiophase stage can be reverted to typical trophophase growth by the addition of inorganic phosphate, suggesting the controlling role of inorganic phosphate in repression and derepression of secondary metabolic and primary metabolic activity of the culture. With a soya peptone-glucose medium, the maximum rate of candicidin production could be maintained and extended for a considerable length of time by controlling the culture pH at 8.0, using glucose to adjust the pH during the later stages of a batch fermentation. Carrying out fermentations in this way has given candicidin yields up to 4 g/liter.

Specific inhibition of candicidin biosynthesis by the lipogenic inhibitor cerulenin

Cerulenin, an inhibitor of fatty acid synthesis, inhibits specifically the biosynthesis of the polyene macrolide candicidin by resting cells of Streptomyces. 50% inhibition was achieved with a cerulenin concentration of 1.5 mug/ml. Cells in which candicidin synthesis was inhibited for 10 h remained capable of candicidin synthesis after removal of the inhibitor. Cerulenin inhibits specifically the incorporation of [14C] propionate into candicidin but does not affect total protein or RNA synthesis. The uptake of [14C] propionate was not inhibited under conditions which totally prevented the incorporation of propionate into candicidin. Incorporation of p-amino[14C] benzoic acid NH2 [14C] BzO- into the aromatic moiety of candicidin was also inhibited by cerulenin. The inhibitory action of cerulenin was not reversed by exogenous fatty acids. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, it is concluded that the polyene macrolide candicidin is synthesized via the polyketide pathway by condensation steps similar to those occurring in fatty acid biosynthesis.

Production of Polyene Macrolide Antibiotics

Publisher Summary This chapter discusses the production of polyene macrolide antibiotics. The polyene macrolides form a subdivision of the macrolide antibiotics containing hydroxylated macrocyclic lactone rings and usually one or more sugars. Biogenetically the macrolides are a homogeneous group, being synthesized from acetate and propionate via the polyketide pathway. The macrolide antibiotics are divided in two subgroups: polyene antifingal antibiotics and nonpolyene antibacterial antibiotics. The polyene subgroup has a system of conjugated double bonds, or chromophore, in the macrolactone ring. This results in an amphipathic molecule containing both a rigid planar lipophilic portion and a flexible hydrophilic polyhydroxylated region. A new class of nonpolyene antifungal macrolides, the axenomycins, with large 34-member lactone rings has been reported. This group appears to be closely related in structure and biological activity to the polyene macrolides in spite of their nonpolyene character. Although purification of polyene macrolide antibiotics is difficult because of their low solubility and instability to heat and light, considerable progress has been achieved in recent years in the determination of the complex chemical structure of these compounds by utilizing sensitive analytical methods, such as electron impact and field desorption mass spectrometry, proton magnetic resonance, and X-ray structure analysis of single crystals for absolute configuration determination.

NEW ANTIBIOTIC PIGMENTS RELATED TO FUSARUBIN FROM FUSARIUM SOLANI (MART.) SACC.

A cholesterol-decomposing fungus, Fusarium solani (Mart.) SACC. strain PP 96, was found to produce several different naphthaquinone pigments in a glycerol-mineral salts medium. Three novel compounds structurally related to fusarubin were isolated by chloroform extration followed by silicic acid column chromatography and preparative thin-layer chromatography. The purified compounds were found to have relatively low activity against bacteria, yeasts and filamentous fungi.

Biosynthesis of candicidin by phosphate-limited resting cells ofStreptomyces griseus

SummaryA phosphate-limited resting cell system ofStreptomyces griseus in a synthetic medium has been developed in which biosynthesis of the polyene macrolide, candicidin, is linear for at least 36 h without cell growth. Glucose and to a lesser degree sucrose, but not lactose, support antibiotic synthesis. Glucose is utilized at a constant rate for antibiotic synthesis without affecting mycelial dry weight. Acetate and propionate, the building units of the macrolide aglycone, stimulate candicidin biosynthesis in cultures supplemented with glucose but do not support its synthesis in the absence of glucose. Maximal stimulation of candicidin biosynthesis was produced by 40 mM propionate or 250 mM acetate. The biosynthetic intermediate, methyl malonate, and the analog, 1-propanol, were more stimulatory than propionate at the same concentration.

Quantitative thin-layer spectrodensitometric determination of the components of polyene macrolide antibiotic complexes

Abstract A direct spectrodensitometric method for quantitating the components of polyene macrolide complexes after separation by thin-layer chromatography is described. Resolution of the components of the candidin and candihexin complexes was good up to 2.5 and 10 μg/spot, respectively. The peak areas were linear with the amount spotted up to the same levels. Maximum peak areas for the components of the candidin and candihexin complexes were obtained using light wavelengths of 360 and 340 nm, respectively. Spotting errors rather than instrumental parameters were responsible for the variance of repeated determinations. Minimal relative standard deviation values were found at intermediate concentration levels in the linear range.

Candihexin Polyene Macrolide Complex: Physicochemical Characterization and Antifungal Activities of the Single Components

The candihexin complex produced by a natural mutant of Streptomyces viridoflavus has been resolved into eight components. At least six of them, having hexaene chromophores, can be separated directly by thin-layer chromatography. The four major components, candihexins A, B, E, and F, account for about 90% of the total complex. They have been isolated and purified to homogeneity. All the major components were found to have macrolide lactone rings with all-trans hexaene chromophores. Candihexins A and B have compositions and molecular weights similar to candidin, the heptaene polyene macrolide produced by the parent S. viridoflavus, whereas candihexins E and F show smaller molecular weights and rather different elementary compositions. Candihexins A and B yield the amino sugar mycosamine upon hydrolysis, but no sugar was found in hydrolysates of candihexins E and F. Candihexins A and B have high antifungal activity against yeasts and filamentous fungi. The amino sugar-lacking candihexins E and F show no antifungal activity at levels of 100 μg/ml. Images

Sequence of Biosynthesis of the Components of the Polyene Macrolides Candidin and Candihexin: Macrolide Aglycones as Intracellular Components

A sequential formation of the single components of the polyene macrolide candidin complex (heptaene) has been found. In addition to the three components occurring in the candidin complex at the end of the fermentation, two other “early” all-trans heptaene components have been characterized. They exist only during the phase of active biosynthesis of candidin. Two of the components of the polyene macrolide candihexin complex (hexaene) that have been described as lacking amino sugar were the only intracellular (mycelium-associated) components observed under conditions in which no extracellular polyene remained attached to the producing cell. The results indicate that glycosylation of the macrolide ring takes place during the secretion process.