Antifungal antibiotic of the basidiomyceteOudemansiella mucida

The developmental cycle of the fungus Oudemansiella mucida, the producer of a new antifungal antibiotic, was found to be controlled by the mechanism of homogenic tetrapolar incompatibility; under our conditions, the cycle took about 12 weeks to completion. Optimum conditions for a laboratory-scale fructification were investigated. Normal fruiting body formation required sufficient illumination, temperatures below 20 degrees C, and relative humidity in excess of 70%. Flask-grown fruiting bodies did not differ from the naturally occurring ones. The basidiospores of the produced fruiting bodies yielded the reference monokaryons necessary for physiological, cytological and genetical studies.     

Antifungal antibiotic of the basidiomyceteOudemansiella mucida

Nuclear ratios were studied in terminal and subterminal cells of various mycelia of the basidiomycete Oudemansiella mucida, the producer of the antifungal antibiotic mucidin (MuciderminR Spofa). The dikaryon, the monokaryon, and the mucidin-producing strain that had been cultivated for a long time under submerged conditions were compared. Dedikaryotization was found to have taken place in the producing strain. The originally dikaryotic culture with characteristic clamp connections on the mycelium and with two nuclei in every hyphal cell lost permanently the clamp connections, probably owing to continuous intense agitation. The hyphae contained solely mononuclear cells. Mating with a compatible monokaryon yielded a dikaryon capable of normal fructification.     

Lipids in fruiting bodies of the basidiomyceteOudemansiella mucida

Dried fruiting bodies of the basidiomyceteOudemansiella mucida contain 29.1% total lipids. Their qualitative analysis revealed the presence of mono-, di-, triglycerides, sterols, free fatty acids and sterolesters. Quantitatively most significant were triglycerides (37.9%) and free fatty acids (29.7%). The phospholipid fraction contained phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid. Gas chromatography showed the presence of a broad spectrum of fatty acids. The ratio between the neutral and polar fractions was 6: 1, both having linoleic acid as the main component.     

Soluble sugars in the mycelium of the basidiomyceteOudemansiella mucida

Quantitative gas chromatography was used to determine soluble neutral sugars in an extract of the fungusOudemansiella mucida grown on a synthetic glucose medium. Apart from the usual fungal sugar components,viz. trehalose,d-glucose,d-mannitol,d-arabinitol, glycerol and inositol, the 6-day-old mycelium containedd-arabino-2-hexosulose (d-glucosone). In the period of maximum growth, this aldoketose was the predominant monosaccharide (3.4 % mycelial dry weight).     

Metabolism of aromatic acids in the antibiotic-producing basidiomyceteOudemansiella mucida

Aromatic acids were determined in the mycelium and fermentation medium ofOudemansiella mucida. Coumaric acids (bothm- andp-),p-hydroxybenzoic acid (salicylic acid) and benzoic acid were found to predominate in the mycelium. Phenylacetic acid represents the main component in the medium. Phenylalanine ammonia-lyase catalyzing conversion of phenylalanine to cinnamie acid which is further metabolized to benzoic acid was detected in the mycelium. The results are discussed with respect to the synthesis of the antibiotic mucidin.     

Effect of tryptophan on the production of mucidin in cultures of basidiomyceteOudemansiella mucida

Addition ofL-tryptophan to cultures of the basidiomyceteOudemansiella mucida brought about a pronounced increase of production of the antibiotic mucidin. The highest increase was reached in the presence of 0.15–0.20 % tryptophan and after its addition to a 1-d culture. The methyl ester of tryptophan exhibited the same effect. Mycelium growing during the initial phases in the presence of tryptophan synthesized mucidin powerfully during later phases of the fermentation. Part VII of the series Antifungal antibiotic of the basidiomyceteOudemansiella mucida; part VI:Folia Microbiol. 27, 35 (1982).     

Composition of lipids and production of mucidin in a submerged culture of the basidiomyceteOudemansiella mucida

Mycelial lipids of the submerged culture ofOudemansiella mucida contain acylglycerols, free and esterified sterols. Free fatty acids are not present. Development of the culture is associated with an increased content of unsaturated fatty acids and, on the contrary, with a decreased content of saturated fatty acids. Content of total lipids depends on age of the culture and is inversely related with production of the antibiotic mucidin.     

Glucose-2-oxidase activity and accumulation of D-arabino-2-hexosulose in cultures of the basidiomyceteOudemansiella mucida

Submerged cultures of the basidiomyceteOudemansiella mucidd, strain III, accumulate D-arabino-2-hexosulose. The maximum yields during cultivations in shaker flasks or in a laboratory fermentor are 6–12 and 15 mg/ml, respectively (20–50 % conversion of substrate glucose). The accumulation is transient, the aldoketose being again utilized after glucose exhaustion. Its production is stimulated by fluoride ions. The enzyme responsible for the C₍₂₎-specific oxidation ofd-glucose acts as an intracellular oxidase with a maximum activity in the exponential phase of growth.d-arabino-2-Hexosulose was also detected in the cultivation medium of the wood-rotting fungiPleurotus ostreatus, Laetiporus sulphureus, andPhellinus abietis. Part III of the series Enzymatic activity of Basidiomycetes; part II:Folia Microbiol. 13, 334 (1968).     

Biosynthesis of fatty acids and sterols in relation to the antibiotic formation inOudemansiella mucida

The production of mucidin by the basidiomyceteOudemansiella mucida was negatively influenced by the application of D-glucitol as the main carbon source, the effect being independent of the growth rate of the mycelium. The rate of fatty acid synthesis was measured by incorporation of 1-¹⁴C-acetate. After 8 days of cultivation, the amount of fatty acids was approximately half that synthetized during cultivation on glucose. The specific rate of incorporation reached its maximum after seven days of cultivation. Incorporation of 2-¹⁴C-mevalonate into sterols was the same under the two sets of cultivation conditions. Acetate units from the degraded fatty acids are probably also utilized for antibiotic synthesis.     

Mucidin-nonproducing mutants ofOudemansiella mucida

Mutants ofOudemansiella mucida, blocked in the biosynthesis of the antibiotic mucidin, were obtained at a 0.28 % frequency after the application of N-methyl-N’-nitro-N-nitrosoguanidine (MNG) to basidiospores under conditions leading to 0.5–5.0 % survival rates. Loss of antibiotic activity was in most isolates accompanied by a decrease in mycelium growth rate and a suppression of dikaryotizing and fructification ability. Recombination analysis of two stable mutants revealed that the block in mucidin synthesis is the result of mutation in the same chromosomal gene(muc). In contrast to the action of MNG, UV-irradiation leads neither to the loss of biosynthetic activity nor to any morphological change.     

Viability of basidiospores ofAgrocybe cylindracea andOudemansiella mucida and antibiotic activity of monosporic isolates in the course of 15 years of storage

Basidiospores of Agrocybe cylindracea were found to be viable for 12 years, those of Oudemansiella mucida for 6 years. The ability of monosporic isolates from these spores to produce antibiotics has been partly preserved.     

Labdane diterpene derivatives from Holwaya mucida

Clumps of white crystals present in 40-day-old malt agar cultures of Holwaya mucida were isolated as long white needles by crystallization from ethanol following short extraction with chloroform. The levorotary compound ([] ₂₈₉ ²¹ =-193.8°) was recognized as a -lactone (C₁₇H₂₀O₅) by infrared and mass spectrometry. It was identified as 7-methoxy-3a, 10b-dimethyl-1, 2, 3, 3a, 5a, 7, 10b, 10c-octahydro-4H, 9H-furo[2, 3, 4 : 4, 5]naphtho[2, 1-c]pyran-4, 9-dione, a labdane-derived compound known as antibiotic LL-Z1271. Preparative thin-layer chromatography of the mother liquor afforded 2 minor metabolites. One was identified as LL-Z1271, the demethylated analogue of LL-Z1271. The other one named LL-Z1271, was recognized as a compound related to and : its structure could not be fully elucidated. H. mucida (anamorph: Crinula calciiformis) has no taxonomic relationship with two other LL-Z1271 producing species viz. Acrostalagmus sp. (= Acremonium cf. atrogriseum) and Oidiodendron truncatum.     

Effect of glucitol on the production of mucidin inOudemansiella mucida

When studying the biosynthesis of mucidin under production (glucose as the main carbon source) and non-production (glucitol as the main carbon source) conditions it could be shown that the producer,Oudemansiella mucida, utilizes glucitol both for growth and for mucidin biosynthesis. However, the production of mucidin is 10 times ower than on glucose. When the culture was preincubated on glucose and transferred to non-production conditions the negative effect of glucitol could not be demonstrated. Biosynthesis of mucidin is influenced by the used carbon source already at an early stage of the cultivation     

Preparation,reversion, mutagenesis and intra-species fusion of protoplasts ofOudemansiella mucida

A technique was developed for preparation and reversion of protoplasts of the mucidin-producing fungusOudemansiella mucida. The protoplasts can be obtained in sufficient amounts from an exponentially growing young mycelium treated with snail digestive juice in the presence of an osmotic stabilizer. The resulting protoplasts readily regenerate their cell walls and, at a frequency of 10-20%, reverse into mycelium. InO. mucida, which does not form asexual spores, protoplasts are a suitable starting material for mutagenesis. Auxotrophic and higher-producing mutants were obtained in this way. Fusion of protoplasts of two compatible monokaryotic auxotrophic isolates, induced by polyethylene glycol, yielded nutritionally complemented dikaryotes forming clamp connections and producing mucidin.     

Use of protoplasts in the improvement of filamentous fungi I. Mutagenization of protoplasts ofOudemansiella mucida

Summary The mutagenic action of ultraviolet light and of N-methyl-N-nitro-N-nitrosoguanidine (NTG) on protoplasts of the basidiomyceteOudemansiella mucida has been evaluated. Since the fungus does not form imperfect spores, we tested the possibilities of using protoplast mutagenesis for improving mucidin-producing strains ofOudemansiella mucida. Protoplasts ofOudemansiella mucida proved to be a convenient starting material for strain improvement and their mutagenization enabled us to obtain not only higher-producing strains, but also auxotrophic mutants, which were used for protoplast fusion experiments.