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.

     

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.     

Preliminary studies on the carcinogenic effects of Penicillium roqueforti toxin (PR toxin) on rats

Preliminary results on the carcinogenic activity of a mycotoxin of Penicillium roqueforti (PR toxin) are reported. A squamous epithelioma and a uterine sarcoma were histologically confirmed in 2 of 10 albino rats fed PR toxin in a relatively short time (449 and 551 days respectively). Only an adenocarcinoma was histologically proven within a control group of 10 rats in a significantly longer (931 days) time span.     

Secondary metabolites resulting from degradation of PR toxin by Penicillium roqueforti.

PR toxin is a secondary metabolite of the fungus Penicillium roqueforti. It is lethal to rats, mice, and cats. Usually, the amount of PR toxin in the culture medium decreases from its maximum on day 15 to zero within 3 to 4 days. We found that two were secondary metabolites produced in the culture medium of this fungus while the production of PR toxin was decreasing. We isolated and purified the two compounds in pure and colorless crystalline form. On the basis of elemental analysis and mass, 1H and 13C nuclear magnetic resonance, infrared, and UV spectroscopies, the two compounds were identified as PR-imine (C17H21O5N) and PR-amide (C17H21O6N). The structures of both compounds and of PR toxin (C17H20O6) were closely related, and the peak production of PR toxin appeared earlier than those of PR-imine and PR-amide. Moreover, PR toxin was transformed to PR-imine when PR toxin was incubated with the culture medium on a given culture day. Thus, we propose that PR toxin is degraded into PR-imine and PR-amide in the culture medium of P. roqueforti.     

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.     

Induction of cross-links between DNA and protein by PR toxin,a mycotoxin from Penicillium roqueforti

PR toxin, a mycotoxin from Penicillium roqueforti, induces DNA—protein cross-links in chromatin of both cultured cells and isolated rat-liver nuclei. The presence of the aldehyde group in the PRT molecule is required for the induction of cross-linking; methylene bridges between nucleic acid and protein are presumably involved in the complex formation. The role of other functional groups of PR toxin is discussed.     

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.     

Survey of the genus penicillium for tremorgenic toxin production

A preliminary survey of the genusPenicillium reveals that production of a trem-orgenic toxin is confined to certain species of the Subsection Fasciculata, Section Asymmetrica. The most intense toxin synthesis was noted in strains ofP. crustosum, P. cyclopium, P. grannulatum, andP. palitans.

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Zusammenfassung Eine vorläufige Übersicht der GattungPenicillium zeigt, daß die Erzeugung eines tremorgenischen Toxins an gewisse Arten der Subsektion Fasciculata, Section Asymmetrica beschränkt ist. Die kräftigste Toxinsynthesis war inP. crustosum, P. cyclopium P. granulatum, undP. palitans beobachtet.

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This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Isolation, Purification, and Characterization of the PR Oxidase from Penicillium roqueforti

The PR oxidase, an extracellular enzyme, involved in the conversion of PR toxin into PR acid, was purified from the culture broth of Penicillium roqueforti ATCC 48936. The enzyme has a pI of 4.5 and a molecular mass of approximately 88 kDa, and it is a monomer. The optimum pH for this enzyme is ca. 4.0, and the optimum temperature is 50°C.     

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.     

Studies on the inhibition of bacterial macromolecule synthesis by roquefortine,a mycotoxin from Penicillium roqueforti

Summary The inhibitory effect of roquefortine, a secondary metabolite of Penicillium roqueforti, on bacterial protein, RNA and DNA synthesis was studied. Similar results were obtained in colorimetric measurements and in studying the incorporation of radioactive precursors. They show that RNA synthesis was most significantly affected by roquefortine. Inhibition of protein and DNA synthesis was less pronounced and might be a result of primary inhibition of RNA synthesis.Abbreviations RNA ribonucleic acid - DNA desoxyribonucleic acid - DMSO dimethylsulfoxide - Cpm counts per min - ATCC American Type Culture Collection