Two Penicillium camembertii mutants affected in the production of cyclopiazonic acid

Penicillium camembertii was mutated and screened for cyclopiazonic acid-negative mutants. With a simple and rapid mini-extraction method for detection of cyclopiazonic acid production, we were able to isolate two strains which were affected in the production of this metabolite. One strain had completely lost the ability to synthesize detectable amounts of this secondary metabolite, whereas the other mutant produced 50 to 100 times less cyclopiazonic acid than the wild type. Also, the former strain had a changed morphology compared with the wild type. This morphological alteration appears to be coupled to the inability to produce cyclopiazonic acid because morphological revertants were able to synthesize cyclopiazonic acid to a level comparable to the wild type. The second mutant accumulated a new metabolite which was detectable by two-dimensional thin-layer chromatography. This new metabolite, however, appears not to be a direct precursor of cyclopiazonic acid.     

The production of cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatoxins by Aspergillus flavus as affected by water activity and temperature on maize grains

The combined effects of water activity (a_w) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid — CPA) and Aspergillus flavus (CPA and aflatoxins — AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P 0.001) between these factors and mycotoxin production. The minimum a_w/temperature for CPA production (2264 ng g^–1 P. commune, 709 ng g^–1 A. flavus) was 0.90 a_w/30 °C while greatest production (7678 ng g^–1 P. commune, 1876 ng g^–1 A. flavus) was produced at 0.98 a_w/20 °C. Least AF (411 ng g^–1) was produced at 0.90 a_w/20 °C and most (3096 ng g^–1) at 0.98 a_w/30 °C.     

Effect of some nutrients on the production of cyclopiazonic acid by Penicillium verrucosum var. cyclopium

Summary The effect of some sugars in different concentrations and some nitrogeneous organic constituents in 2% yeast extract (basal medium) on production of cyclopiazonic acid (CA) by Penicillium verrucosum var. cyclopium was studied at room temperature. Maximum CA production was observed after 14 days in a medium containing 2% yeast extract +2.5% sucrose. Ammonium lactate had a negative effect on the production of CA by the test culture. Nitrogeneous organic constituents such as peptone and tryptone did not enhance the yield of CA in the medium. After an initial drop in the pH, a general increase in pH was observed as the incubation time increased. Curdled milk was also found to be a suitable medium for the production of CA by the mold culture.Recipient of Deutscher Akademischer Austauschdienst (DAAD) fellowship 1984–1985 (National Dairy Research Institute, Karnal-132 001, India)     

Crystallization and characterization of monoacylglycerol and diacylglycerol lipase from Penicillium camembertii.

A new lipase from Penicillium camembertii U-150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalin-A-Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalin-A-Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. All four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase.     

Aflatoxin and cyclopiazonic acid production by a sclerotium-producing Aspergillus tamarii strain.

The production of aflatoxins B1 and B2 by Aspergillus tamarii (subgenus Circumdati section Flavi) is reported for the first time. The fungus was isolated from soil collected from a tea (Camellia sinensis) field in Miyazaki Prefecture, Japan. Three single-spore cultures, NRRL 25517, NRRL 25518, and NRRL 25519, were derived from subcultures of the original isolate 19 (MZ2). Each of these single-spore cultures of A. tamarii produced aflatoxins B1 and B2 and cyclopiazonic acid, as well as black, pear-shaped sclerotia. The demonstration of aflatoxin production by A. tamarii is examined in connection with A. tamarii phylogenetic relationships, chemical ecology, and potential use in food fermentations.     

The effect of lactate addition on the growth of Penicillium camembertii on glutamate

The effect of an additional carbon source, lactate, on Penicillium camembertii growth on glutamate as both carbon and nitrogen sources was examined. Glutamate (and lactate) was present in excess in both media. Throughout the whole culture, similar growth time-courses were recorded on both media, indicating the absence of a lactate effect on growth. During the first part of growth, corresponding to an increasing amount of viable biomass, the rate of glutamate consumption remained high, as well as the related ammonium production, indicating its use as a carbon source in addition to being nitrogen source. The low growth rates recorded during the last part of growth resulted in low glutamate consumption, while lactate consumption continued mainly by a maintenance mechanism for the energy supply. A clear differentiation appeared therefore between the carbon source and the energy source: glutamate was mainly used as C source (and N source) for biosynthesis, while lactate was mainly assimilated for energy supply. Carbon and nitrogen yield examinations confirmed this result. Indeed, the C/N ratio found for P. camembertii cellular material (8.14) was about twice that of glutamate (4.29). From this, about half of the available nitrogen was used for biomass formation during growth on glutamate-lactate based medium, as experimentally confirmed (constant yield nitrogen from biomass on nitrogen from glutamate was found (0.49), while the excess nitrogen was released as ammonium). The constant and close to unit (0.99) yield carbon from CO2 on carbon from lactate, also recorded during growth on glutamate-lactate based medium, confirmed that lactate was mainly used as an energy source.     

Mutagenicity of tetramic mycotoxin cyclopiazonic acid.

Cyclopiazonic acid was shown to be mutagenic to Salmonella typhimurium TA98 and TA100 in the presence of metabolic activation. The activity of cyclopiazonic acid in the presence of aflatoxin B1 was studied in complete factorial experiments with strain TA98. Both mycotoxins produced significant mutagenic activity and in combination. The activity in combination appeared to be additive rather than synergistic. The specific activity of cyclopiazonic acid was estimated to be approximately 140 revertants per mu mol in strain TA98.     

Mycophenolic acid production by Penicillium brevicompactum in two media

Penicillium brevicompactum produces mycophenolic acid as it grows vegetatively, not only on a simple medium where growth is slow but also on a richer medium where growth is less restricted. The implications of this finding on the association of fungal secondary metabolism with the idiophase in liquid and solid culture are discussed.     

Production of cyclopiazonic acid by Aspergillus flavus Link.

Production of cyclopiazonic acid by Aspergillus flavus is reported for the first time. A procedure for its production by agitated solid substrate fermentation on red wheat is described along with the isolation procedure and physical and chemical properties of this indole derivative. The compound has been found to exert antibacterial activity.     

Neurochemical effects of cyclopiazonic acid in chickens

Chickens dosed (per os) with cyclopiazonic acid (CPA) at 0.5, 5.0, and 10 mg/kg body weight showed significant (P less than or equal to 0.05) increases in brain dopamine and serotonin concentrations 96 hr after dosing. The increases coincide with significant decreases in homovanillic acid and subtle increases of dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid concentrations. The elevated dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid concentrations may be related to the elevated concentrations of dopamine and serotonin, respectively. The observed changes in neurotransmitter/metabolite concentrations 96 hr after dosing parallel elimination of CPA from the birds skeletal muscles; however, they do not correlate with the significant weight losses in these birds at 48 and 96 hr after dosing. The brain weights of the treated birds were statistically insignificant from their respective controls, although increases in brain weight-body weight ratio within treatments and with time correlated with CPA toxicity. No significant changes were observed in dopamine, dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid concentrations among the treatments at 3, 24, and/or 48 hr after dosing.     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Production of cyclopiazonic acid by Aspergillus tamarii Kita.

Production of the mycotoxin cyclopiazonic acid by Aspergillus tamarii Kita is reported for the first time. Examination of 23 isolates of the fungus showed that 22 produced the toxin under the culture conditions utilized.     

Effect of fatty acids on cellulase production by Penicillium funiculosum and its mutants

Summary Penicillium funiculosum and its mutants namely BU-36 and N-4 responded differently to the addition of fatty acids. Addition of 0.1% oleic, linoleic, and linolenic acids resulted in significant increase in extracellular exo-glucanase and -glucosidase in wild and N-4 strains, whereas no appreciable increase was noticed in BU-36. However, BU-36 showed positive response with 0.1% palmitic and stearic acids. In all the strains, the addition of different fatty acids did not have any effect on endoglucanase activity. Our results indicate that fatty acids do have a role in the release of the cell-bound cellulolytic enzymes.NCL Communication No. : 4113     

Abnormal amino acid metabolism in mutants of Saccharomyces cerevisiae affected in the initiation of sporulation

Amino acid metabolism was examined during sporulation in a wild-type strain of Saccharomyces cerevisiae and in a mutant homozygous for the spd1 mutation (derepression of sporulation). In the wild-type initiation of sporulation involves a rise in intracellular glutamate. In the derepressed mutant there was no further increase of intracellular glutamate: glutamate was already present at a greater concentration than in the wild-type. These elevated glutamate levels are apparently due to reduction in the activity of 2-oxoglutarate dehydrogenase. One consequence of the elevated glutamate levels in diploids homozygous for the spd1 mutation is the production of considerable amounts of glycyl-L-proline, glutathione and saccharopine.     

Kinetic characterization in batch and continuous culture of Escherichia coli mutants affected in phosphoenolpyruvate metabolism: differences in acetic acid production

The growth kinetics of an Escherichia coli wild type strain and two derivative mutants were examined in batch cultures and in glucose-limited chemostats. One mutant (PB12) had an inactive phosphotranferase transport system and the other (PB25) had interrupted pykA and pykF genes that code for the two pyruvate kinase isoenzymes. In both batch and continuous culture, important differences in acetic acid accumulation and other metabolic activities were found. Compared to the wild type strain, we observed a reduction in acetic acid accumulation of 25 and 80% in PB25 and PB12 strains respectively, in batch culture. Continuous culture experiments revealed that compared to the other two strains, PB25 accumulated less acetic acid as a function of dilution rate. In continuous cultures, oxidoreductase metabolic activities were substantially affected in the two mutant strains. These changes in turn were reflected in different levels of biomass and CO₂ production, and in oxygen consumption.     

Inhibition of translation by the fungal toxin cyclopiazonic acid

Cyclopiazonic acid is a toxic metabolite of fungal origin that inhibits protein synthesis in intact HeLa cells and eukaryotic cell-free systems. It has been shown that cyclopiazonic acid blocks the GTP- and EF-1-dependent binding of (³H)Phe-tRNA to 80S ribosomes. Moreover the translocation of N-Ac-(³H)Phe-tRNA by 80S ribosomes that takes place in the presence of EF-2 and GTP is also halted by cyclopiazonic acid. It is concluded that this drug affects a ribosomal site involved in the alternative interaction of elongation factors EF-1 and EF-2.