Ultraviolet-B Lethal Damage on Pseudomonas aeruginosa

A secondary metabolite different from PR-imine and PR-amide was produced in the liquid (YESC) and solid (buckwheat) culture medium of Penicillium roqueforti. We isolated and purified the compound in pure and colorless crystalline form. On the basis of elemental analysis, mass, ¹H and ¹³C nuclear magnetic resonance, infrared, and UV spectroscopy, the compound was identified as PR-acid (C₁₇H₂₀O₇). The structures of PR-acid and PR toxin (C₁₇H₂₀O₆) are closely related. Moreover, we discovered that PR-acid disappeared concurrently with the PR toxin in the culture medium. Thus, we postulate that PR toxin is degraded to PR-acid in the culture of P. roqueforti.     

Potentiation effect of corn extract on the production of eremofortin C, EC oxidase, and PR toxin by Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Their structures are similar and differ only by an alcohol and an aldehyde group at the C-12 position. EC has been demonstrated to be the precursor of PR toxin, and EC is transformed to PR toxin by EC oxidase. These two compounds and EC oxidase are secreted by P. roqueforti in the culture medium, which is usually composed of 15% sucrose and 2% yeast extract. Recently, we discovered that the addition of corn extract to this medium increased the production of EC and PR toxin and the activity of EC oxidase in a coordinative manner. In a time-course study, we found that the peak yield of EC and PR toxin and the maximum activity of EC oxidase in the culture medium containing 7.5% sucrose, 1% yeast extract, and 20% corn extract were increased 6.2, 4.6, and 4.7-fold, respectively, as compared with those obtained in the medium without corn extract. Moreover, corn extract increased the production of EC and PR toxin and the activity of EC oxidase by P. roqueforti in a dose-dependent manner. On the other hand, when the concentrations of sucrose and yeast extract were increased while fixing the ratio of corn extract, we found that the levels of EC and PR toxin and the enzyme activity were decreased concomitantly. We thus conclude that corn extract can enhance the production of EC, PR toxin and EC oxidase by P. roqueforti when grown in a minimal medium and that the potentiation effect of corn extract is suppressed when the fungi are grown in a rich medium.     

Isolation and Some Properties of the Enzyme That Transforms Eremofortin C to PR Toxin

PR toxin and eremofortin C are secondary metabolites of Penicillium roqueforti. The chemical structures of these two compounds are closely related to each other and differ only by an aldehyde and an alcohol group at the C-12 position. In an effort to better understand the biosynthesis of PR toxin, we discovered the enzyme of P. roqueforti that is responsible for the transformation of eremofortin C to PR toxin. The maximum activity of the enzyme in the culture medium was found to occur on day 13, which corresponded to the maximal production of PR toxin in the medium. The enzyme was isolated and purified from the culture medium and the mycelium of the fungus, respectively, through a procedure involving ammonium sulfate fractionation and DEAE-cellulose chromatography. The specific activity increased 20- and 8-fold, respectively, and the yield was 33.3 and 21.6%, respectively, for the enzyme from the medium and mycelium. The optimal pH for the enzyme reaction was ca. pH 5.6. The enzyme reaction was temperature dependent. The rates followed a linear time course when it catalyzed the transformation at 30°C and decayed with time when reacted at higher temperatures. At 100°C, the enzyme activity was completely lost. The K_m and V_max of the enzyme as determined at 30°C were 0.02 mM and 4.0 μmol/min per mg, respectively. The molecular weight of the enzyme was estimated by gel filtration on a high-pressure liquid chromatography I-250 protein column to be ca. 40,000.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Study of conditions of production of roquefortine and other metabolites of Penicillin roqueforti.

Experiments to determine optimum yields of roquefortine, isofumigaclavine A, and PR toxin, metabolites from Penicillum roqueforti Thom, were performed. Four strains, isolated from blue cheese, and five liquid media were evaluated, although not all permutations were studied. Sucrose (15%)-yeast extract (2%) was the medium chosen for time-course studies at 25 and 15 degrees C using one favorable strain. At 25 degrees C, maximum estimated yields of roquefortine were about 100 mg/liter in the mycelium by 16 days, and no subsequent degradation of this alkaloid was observed. On the other hand, production of PR toxin in the medium peaked at 770 mg/liter at 21 days. At 15 degrees C, yields of roquefortine and PR toxin after 49 days were 60 to 70% of the maximum yields obtained at 25 degrees C. However, about three times more isofumigaclavine A (up to 11 mg/liter) was formed in the mycelium at 15 degrees C than at 25 degrees C. All four strains of P. roqueforti procedure both roquefortine and PR toxin on the sucrose-yeast extract medium at 25 degrees C; isofumigaclavine A was detected in all but one strain grown on this medium.     

Measurement of mercury methylation in lake water and sediment samples

The production of various eremophilane-type sesquiterpenes by Penicillium roqueforti strains has allowed us to propose a biochemical pathway for PR toxin synthesis. A time-course study of P. roqueforti metabolite production by high-performance liquid chromatography was performed to check this hypothetical pathway. The results obtained suggested that eremofortin C was the direct precursor of PR toxin in the P. roqueforti cell. Attempts to determine the amount of PR toxin in the mycelium failed. It was shown that the absence of PR toxin in mycelium was due to its instability during the extraction procedure.     

PR toxin production in different Penicillium roqueforti strains.

Different Penicillium roqueforti strains from the American Type Culture Collection were tested for the production of PR toxin. All four strains were able to produce the toxin on semisynthetic medium at 24 degrees C after certain periods of incubation. The yields were correlated with the pH of the medium. Timing of the harvest influenced both the yield and purification of the toxin.     

PR toxin production in different Penicillium roqueforti strains.

Different Penicillium roqueforti strains from the American Type Culture Collection were tested for the production of PR toxin. All four strains were able to produce the toxin on semisynthetic medium at 24 degrees C after certain periods of incubation. The yields were correlated with the pH of the medium. Timing of the harvest influenced both the yield and purification of the toxin.     

Chemical transformation of eremofortin C into PR toxin.

The natural products of both eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Because the chemical structures of EC and PR toxin are closely related to each other and differ only by a hydroxyl functional group in EC and an aldehyde functional group in PR toxin at the C-12 position, the chemical transformation of EC into PR toxin was investigated. Oxidation with a chromic anhydride-pyridine complex was found to be the most satisfactory method.     

Chemical transformation of eremofortin C into PR toxin

The natural products of both eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Because the chemical structures of EC and PR toxin are closely related to each other and differ only by a hydroxyl functional group in EC and an aldehyde functional group in PR toxin at the C-12 position, the chemical transformation of EC into PR toxin was investigated. Oxidation with a chromic anhydride-pyridine complex was found to be the most satisfactory method.     

Biochemical effects of PR toxin on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of PR toxin, a toxic secondary metabolite produced by certain strains of Penicillium roqueforti, on the membrane structure and function of rat liver mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the toxin was added to the assay system. The respiratory control ratio decreased about 60% and the ADP/O ratio decreased about 40% upon addition of 3.1 X 10(-5) M PR toxin to the highly coupled mitochondria. These findings suggest that PR toxin impairs the structural integrity of mitochondrial membranes. On the other hand, the toxin inhibited mitochondrial respiratory functions. It exhibited noncompetitive inhibitions to succinate oxidase, succinate-cytochrome c reductase, and succinate dehydrogenase activities of the mitochondrial respiratory chain. The inhibitory constants of PR toxin to these three enzyme systems were estimated to be 5.1 X 10(-6), 2.4 X 10(-5), and 5.2 X 10(-5) M, respectively. Moreover, PR toxin was found to change the spectral features of succinate-reduced cytochrome b and cytochrome c1 in succinate-cytochrome c reductase and inhibited the electron transfer between the two cytochromes. These observations indicate that the electron transfer function of succinate-cytochrome c reductase was perturbed by the toxin. However, PR toxin did not show significant inhibition of either cytochrome oxidase or NADH dehydrogenase activity of the mitochondria. It is thus concluded that PR toxin exerts its effect on the mitochondrial respiration and oxidative phosphorylation through action on the membrane and the succinate-cytochrome c reductase complex of the mitochondria.     

Exogenous H(2)O(2) and catalase treatments interfere with Tri genes expression in liquid cultures of Fusarium graminearum

Effect of exogenous H(2)O(2) and catalase was tested in liquid cultures of the deoxynivalenol and 15-acetyldeoxynivalenol-producing fungus Fusarium graminearum. Accordingly to previous results, H(2)O(2) supplementation of the culture medium leads to increased toxin production. This study indicates that this event seems to be linked to a general up regulation of genes involved in the deoxynivalenol and 15-acetyldeoxynivalenol biosynthesis pathway, commonly named Tri genes. In catalase-treated cultures, toxin accumulation is reduced, and Tri genes expression is significantly down regulated. Furthermore, kinetics of expression of several Tri genes is proposed in relation to toxin accumulation. Biological meanings of these findings are discussed.     

辣椒炭疽病菌毒素

辣椒炭疽病菌(辣椒刺盘孢Colletotrichum capsici)在25—28℃的Czapek-Dox培养液中振荡培养时分泌出毒素,这种毒素能引起辣椒叶片形成坏死斑,类似于病原菌侵染形成的症状。辣椒叶煎汁培养液和Czapek-Dox培养液适于C.capsici的生长和产毒,生长适宜温度和范围分别为25℃和pH6—7,在25—28℃,pH6—7的条件下培养滤液毒性最强;光线和通气条件可促进C.capsici的生长和产毒;培养14天的培养滤液毒性最强。培养滤液经丙酮沉淀及离子交换树脂柱和Sephadex G-50柱层析将毒素纯化。实验结果表明该毒素为多聚糖类物质。生物检测结果表明:培养滤液和C.capsici毒素溶液能抑制辣椒、绿豆、豌豆、豇豆种子胚根生长;并能使辣椒幼苗发生萎蔫,这一作用与辣椒品种抗性有关。     

Penicillium roqueforti PR toxin gene cluster characterization

PR toxin is a well-known isoprenoid mycotoxin almost solely produced by Penicillium roqueforti after growth on food or animal feed. This mycotoxin has been described as the most toxic produced by this species. In this study, an in silico analysis allowed identifying for the first time a 22.4-kb biosynthetic gene cluster involved in PR toxin biosynthesis in P. roqueforti. The pathway contains 11 open reading frames encoding for ten putative proteins including the major fungal terpene cyclase, aristolochene synthase, involved in the first farnesyl-diphosphate cyclization step as well as an oxidoreductase, an oxidase, two P450 monooxygenases, a transferase, and two dehydrogenase enzymes. Gene silencing was used to study three genes (ORF5, ORF6, and ORF8 encoding for an acetyltransferase and two P450 monooxygenases, respectively) and resulted in 20 to 40% PR toxin production reductions in all transformants proving the involvement of these genes and the corresponding enzyme activities in PR toxin biosynthesis. According to the considered silenced gene target, eremofortin A and B productions were also affected suggesting their involvement as biosynthetic intermediates in this pathway. A PR toxin biosynthesis pathway is proposed based on the most recent and available data.

     

Penicillium verruculosum SG: a source of polyketide and bioactive compounds with varying cytotoxic activities against normal and cancer lines

A newly isolated fungus Penicillium verruculosum SG was evaluated for the production and characterization of bioactive colored secondary metabolites using solid-state fermentation along with their cytotoxic activities against normal and cancer cell lines. Logical fragmentation pattern following column chromatography, thin layer chromatography and liquid chromatography and mass spectrometry of crude culture filtrate of fungus revealed the presence of different polyketide pigments and other bioactive compounds. Cytotoxicity of the selected colored fractions of fungal filtrate containing different compounds revealed IC50 (μg/ml) values ranging from 5 to 100. It was significantly higher in case of orevactaene (5 + 0.44) and monascorubrine followed by pyripyropene (8 + 0.63) against cancer cell line KA3IT. Overall, these compounds considerably showed less toxicity toward normal cell lines NIH3T3, HSCT6, HEK293 and MDCK. XRD of a yellow crystalline compound (224.21 m/z) confirmed its 3-dimensional structure as phenazine 1 carboxylic acid (C13H8N2O2) (broad spectrum antibiotic), and it is first time reported in fungi.     

Mass production of pure gibberellin A1 by Phaeosphaeria sp. L487 and the fungal preparation of [U-13C]gibberellin A1.

The mass production of pure gibberellin A1 (GA1) by shake-culturing Phaeosphaeria sp. L487 was investigated. Its GA1 production was markedly influenced by natural nitrogen sources and NH4NO3. When the fungus was cultured in an 8% glucose-1.5% oatmeal-0.1% NH4NO3-0.5% KH2PO4-0.1% MgSO4 x 7H2O medium for 3 weeks, the amount of GA1 in the culture filtrate was up to ca. 200 microg/ml: the addition of safflower oil to the culture medium two weeks after inoculation prolonged the GA1-production period to produce 300 microg/ml. Further preparation of [U-13C]GA, as a tool for the analysis of a complex of GA1 and its binding protein was attempted by using the fungus. The fungal culture in a [U-13C]glucose-oatmeal medium gave 6 mg of crystalline 13C-enriched GA1. Its 13C-enrichment of ca. 75% and 1J(CC) values were determined by NMR spectrometry.     

Isolation and Partial Characterization of a Mycotoxin from Penicillium roqueforti

Extracts of pure cultures of Penicillium roqueforti isolated from toxic feed samples and of P. roqueforti NRRL 849 were lethal to rats by either intraperitoneal or oral administration. Purification studies guided by this test led to the isolation of a major toxin which showed intraperitoneal and oral median lethal dose values in weanling rats of 11 and 115 mg/kg, respectively. Partial characterization of the crystalline compound, C(17)H(20)O(6), by infrared, ultra violet, PMR, and mass spectroscopy, and by several chemical transformations indicated the presence of three C-methyl substituents plus one acetoxy, one aldehyde, and one alpha,beta-unsaturated ketone group. Two oxygen atoms are present either in epoxide or ether form.     

Effect of culture medium composition on the activity of extracellular lectins of Lentinus edodes

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under the conditions of submerged culture was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and pH of culture medium. The activity of lectin in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C : N ratio, (9.5-12): 1)) on the day 15-18 of culturing at pH 8-9.