Characterization and regulation of new secondary metabolites from Aspergillus ochraceus M18 obtained by UV mutagenesis

UV irradiation of Aspergillus ochraceus NRRL 3174 conidia led to stable mutations in ochratoxin and penicillic-acid pathways. These mutants, especially M18, produced an unexpectedly large number of new metabolites. Two new compounds were purified by TLC and HPLC and their chemical structures were determined. They are 2,10-dimethyl 4-hydroxy-6-oxo-4-undecen-7-yne (1) and 4-(3-methyl-2- butenyl) oxy 1-phenyl acetic acid (2). Compound 1 is very active against Gram-positive bacteria, such as Staphylococcus aureus and Bacillus subtilis, but inactive against Gram-negative bacteria, fungi, and yeasts. However, compound 2 has no antibiotic activity. The production of 1 was generally associated with growth, whereas that of compound 2 was dissociated from growth. The biosynthesis of these 2 metabolites was influenced by the sources of carbon and nitrogen.     

Simultaneous quanitative determination of secondary metabolites ofAspergillus ochraceus by TLC

A simple TLC method for the quanitative determination of mellein, 4-hydroxy-mellein, penicillic acid, ochratoxin A, B, α and β is described. Application of this technique permits metabolic studies of the influence of different factors on the formation ofAspergillus ochraceus metabolites.     

Biosynthesis of ochratoxins by Aspergillus ochraceus.

Shaken liquid fermentation of an isolate of Aspergillus ochraceus showed growth-associated production of ochratoxins A and B, followed by production of a related polyketide diaporthin. Later, between 150 and 250 h, mellein accumulated transitorily. In contrast, shaken solid substrate (shredded wheat) fermentation over 14 days produced mainly ochratoxins A and B (ratio ca. 5:1) in very high yield (up to 10 mg/g). In these systems experiments with 14C-labelled precursors and putative intermediates revealed temporal separation of early and late stages of the ochratoxin biosynthetic pathway, but did not support an intermediary role for mellein. The pentaketide intermediate ochratoxin beta was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin alpha was only biotransformed significantly (4.85%) into ochratoxin A, indicating that chlorination is mainly a penultimate biosynthetic step in the biosynthesis of ochratoxin A. This was supported by poor (1.5%) conversion of radiolabelled ochratoxin B into ochratoxin A. Experiments implied that some ochratoxin B may arise by dechlorination of ochratoxin A.     

Transformation in Aspergillus ochraceus

Mutants (lysine requiring) of Aspergillus ochraceus were kept under starvation conditions for 15 days and finally were treated with DNA of a 40-h-old culture of the wild strain. The donor DNA-treated mutant conidia were then grown on plates containing minimal medium at 28°C for 4 days. The number of transformed cells was estimated by colony counting and hence percentage transformants. The transforming activity of the donor DNA was found to be inhibited by the action of heat and variation of pH, and also varied with the period of starvation and with the concentration of donor DNA.     

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

Biosynthesis of diaporthin and orthosporin by Aspergillus ochraceus

Diaporthin and orthosporin were characterised from the fungus Aspergillus ochraceus D2306. Diaporthin was identified by high-resolution electron impact mass spectrometry and 1H and 13C NMR spectroscopy, from which new spectroscopic assignments were made. Orthosporin was also identified by mass spectrometry and both fungal metabolites are reported for the first time as co-metabolites and also as products of A. ochraceus. The methylation inhibitor ethionine affected production of both diaporthin and orthosporin in spite of no obvious methylation step in the biosynthesis of orthosporin, implying that extracellular orthosporin may arise by de-O-methylation of diaporthin. The biosynthetic origin of diaporthin was demonstrated by incorporation of [1-14C]acetate and [methyl-14C]methionine administered in early idiophase.     

Mycotoxicity of Aspergillus ochraceus to Chicks

Five isolates of Aspergillus ochraceus, obtained from peanuts, were grown separately on sterile, moist corn for 14 days and fed to 1-day-old Babcock B-300 cockerels to evaluate their toxic effects. Two isolates were highly toxic, causing death of all birds during the 1st week of the experiment. Two isolates were moderately toxic, causing severe growth suppression with some deaths occurring throughout the 3-week test period. One isolate had no apparent effect. When the two most toxic isolates (diets) were diluted, survival time increased but severe growth suppression was evident. Postmortem examinations revealed a few small hemorrhages in the proventriculi of birds which died between the 2nd and 5th days. Emaciation, dehydration, and dry, firm gizzard linings were observed throughout the experiment. Extensive hepatic injury consisting of either fatty changes or necrotic foci was the principal microscopic finding. Suppression of bone marrow activity and depletion of lymphoid elements in the spleen and bursa of Fabricius were also found. The severity of the histopathological changes was directly related to the concentration of ochratoxin A in the diets.     

Mellein and 4-Hydroxymellein Production by Aspergillus ochraceus Wilhelm

Mellein and 4-hydroxymellein are isocoumarin compounds produced by Aspergillus ochraceus Wilhelm. They are structurally similar to the dihydroisocoumarin moiety of ochratoxin A, a toxic metabolite of the same fungus, and they possibly have similar biological properties. Production of mellein and 4-hydroxymellein on synthetic media and natural solid substrates was determined. Several carbon and nitrogen sources supported production of these metabolites in stationary culture. Additional zinc and molybdenum increased production of both metabolites in stationary culture, but were not required for maximum production in shaken culture. Mellein and 4-hydroxymellein were produced on yellow corn, but neither was produced on wheat, peanuts, or soybeans.     

Biodegradation of Reactive blue-25 by Aspergillus ochraceus NCIM-1146

The present study dealt with the decolorization and degradation of textile dye Reactive blue-25 (0.1gl(-1)) by mycelium of Aspergillus ochraceus NCIM-1146. Spectrophotometric and visual examinations showed that the decolorization was through fungal adsorption, followed by degradation. Shaking condition was found to be better for complete and faster adsorption (7h) and decolorization (20 days) of dye Reactive blue-25 (100mgl(-1)) as compared to static condition. Presence of glucose in medium showed faster adsorption (4h) and decolorization of dye from bound (7 days) mycelium. FTIR and GCMS analysis study revealed biodegradation of Reactive blue-25 into two metabolites phthalimide and di-isobutyl phthalate.     

An improved 11 alpha-hydroxylation of progesterone by Aspergillus ochraceus TS.

1. The stereospecific hydroxylation of progesterone exclusively to its 11 alpha-hydroxy derivative by Aspergillus ochraceus TS is reported. 2. There is no secondary metabolite (6beta, 11 alpha-dihydroxyprogesterone) formed even when the transformation was attempted with different concentrations of the substrate (200 microgram/ml-200 mg/ml) for prolonged with Zn2+ at a concentration of 50 microgram/ml identical results were obtained. 3. Similar results were also obtained at different pH values of the culture medium.     

Detection and quantification of Aspergillus ochraceus in green coffee by PCR

AIMS: The aim of this study was to detect and quantify DNA of the ochratoxinogenic fungus Aspergillus ochraceus in green coffee and to compare the results with the ochratoxin A content of naturally contaminated samples. METHODS AND RESULTS: A DNA extraction protocol based on a combination of ultrasonification and a commercial kit was tested for the recovery of fungal DNA. PCR and real-time PCR protocols were established for the detection of A. ochraceus. Sensitivity of the PCR was checked by the addition of inoculated green coffee and pure fungal DNA to uncontaminated green coffee samples. The A. ochraceus DNA content of 30 naturally contaminated green coffee samples was determined and compared with the ochratoxin A concentrations. CONCLUSIONS: Aspergillus ochraceus can be rapidly and specifically detected in green coffee by PCR. A positive correlation between the ochratoxin A content and the DNA quantity was established. Significance and Impact of the Study: This work offers a quick alternative to the conventional mycological detection and quantification of A. ochraceus in green coffee.     

Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

利用赭曲霉NG1203羟基化齐墩果酸的研究

考察了利用赭曲霉Aspergillus ochraceusNG1203进行C11α-羟基化齐墩果酸生化反应的条件。得出了菌株摇瓶培养最佳培养基配方(g.L-1):葡萄糖20,玉米浆25,酵母膏3,K2HPO41.5,pH 6.0。菌株培养20 h,加入2 mg.L-1的齐墩果酸利于诱导羟基化酶的产生。菌株在28℃下以150 r.min-1振荡培养24 h,加入底物的乙醇溶液,使转化液中齐墩果酸的初始质量浓度达100 mg.L-1,转化液中乙醇体积分数最终达3%。经96 h转化,齐墩果酸转化率可达到10.12%。通过HPLC1、H NMR和13C NMR分析,结果表明产物为C11α-羟基齐墩果酸。     

Bioconversion of Progesterone by the Activated Immobilized Conidia of Aspergillus ochraceus TS

Progesterone was transformed to its 11α-hydroxy derivative (100% e.e) by the activated immobilized conidia of Aspergillus ochraceus TS. The immobilized preparation retained 79% of free conidial activity. The immobilized conidia, activated by nutrients, exhibited an increase in 11α-hydroxylation, and it was free of the side product 6β, 11α-dihydroxy progesterone. The half life and turnover of immobilized and activated immobilized conidia were 14 and 12 days and 187 and 416 μmoles of the product/g of conidia respectively. The pH and temperature profiles of the free conidia remained unaltered after immobilization and activation. Some germination of conidia inside the matrix owing to incubation with nutrients was detected by scanning electron microscopy. Received: 18 March 1999 / Accepted: 1 July 1999     

Characterization of a novel microsomal glutathione S-transferase produced by Aspergillus ochraceus TS

Purification and characterization of microsomal glutathione S-transferase produced by Aspergillus ochraceus TS are reported. The isozymes are located in microsomes and were active against 1-chloro-2,4-dinitrobenzene, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, trans-4- phenyl-3-buten-2-one,p-nitrobenzyl chloride and bromosulphophthalein. They were inhibited by N-ethylmaleimide and bromosulphophthalein. The GST isozymes produced by Aspergillus ochraceus TS are indistinguishable in respect of their molecular mass both in native and denatured state. The subunit of the purified protein had an apparent M_r of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pl values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.     

Two-Liquid phase reactor studies of 11alpha-hydroxylation of progesterone by Aspergillus ochraceus

The 11alpha-hydroxylation of progesterone by free cells of Aspergillus ochraceus has been examined in a reactor with an aqueous and a natural oil phase. As the proportion of oil is increased, the point of inversion from a continuous aqueous phase is affected by the cell concentration, as is the optimum ratio of the phases for conversion. High oil ratios are not productive because of poor mass transfer to the concentrated cells in the aqueous phase. The cells are stable in the presence of oleic acid for at least 24 h.