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.     

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.     

Ability of casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461

The ability of casamino acids and vitamin-assay casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461 was examined in a medium containing glucose or corn syrup as the carbon source relative to yeast extract supplementation. When glucose or corn syrup served as the carbon source, the presence of yeast extract in the growth medium stimulated gellan production by strain ATCC 31461 on casamino acids. Using vitamin-assay casamino acids as the nitrogen source, the addition of vitamins lowered gellan synthesis by glucose-grown cells regardless of yeast extract supplementation while gellan elaboration by corn syrup-grown strain ATCC 31461 cells could only be increased by supplementing vitamins into medium lacking yeast extract. Independent of carbon source, the absence of yeast extract in the medium reduced biomass production. Biomass production by the strain grown on either carbon source was increased by supplementing vitamins in the medium containing yeast extract.     

Preparation and characteristics of mutant strains of Penicillium funiculosum--producers of glucose oxidase

After the mutagenesis of Penicillium funiculosum with UV light and N-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5-153% higher (in a medium with glucose) and 4-83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutant P. funiculosum NMU95-132 was chosen for further selection work.     

Tea extract as an inexpensive inducer of pectin lyase in Penicillium griseoroseum cultured on sucrose

Extracts of tea, coffee, cocoa, and yeast induced pectin lyase (PL) in Penicillium griseoroseum cultured in a mineral medium with sucrose as the carbon source. PL activity and fungal growth were similar in the treatments with 0.5% tea extract, the highest concentration tested, and 0.03% yeast extract. When tea extract was added singly to the culture medium, P. griseoroseum produced 59% and 17% of the PL activity and mycelial mass, respectively, obtained in a treatment with tea extract and sucrose. These results suggest that the production of the enzyme was not proportional to mycelial growth. No PL was produced in the medium with sucrose and without inducers. The small amounts of pectic substances present in the tea extract could not be responsible for PL induction. PL activity was detected after 12 h of growth in the medium containing sucrose and tea extract added at time zero, and after 48 h of growth when tea extract was added at times 12 and 24 h. Mycelial mass in all treatments was similar after 48 h of incubation. However, the addition of tea extract at time zero increased PL activity by 20–25%. Cyclic AMP at 5 and 10 mM in the culture medium induced 20 and 30%, respectively, of the PL activity obtained with 0.03% yeast extract, suggesting that PL induction brought about by either yeast extract or tea extract might involve the intracellular metabolism of cAMP. Received 22 October 1996/ Accepted in revised form 09 January 1997     

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.     

Identification of cheese-associated fungi using selected ion monitoring of volatile terpenes

The cheese-associated fungi Penicillium commune , P. roqueforti , P. solitum , P. discolor and Aspergillus versicolor have been investigated for production of volatile terpenes for chemical identification, when grown on yeast extract agar. Volatiles were collected by headspace solid-phase microextraction. Selected ion monitoring of four to seven of the most characteristic ions of mainly sesquiterpenes made it possible to identify the fungi to species level within 2 d. In a mixed culture of P. roqueforti and P. commune , inoculated in a ratio of 1000 : 1, volatiles from both fungi could be detected within 3 d, making identification possible.     

Production of chitinolytic enzymes by a strain (BM17) of Paenibacillus pabuli isolated from crab shells samples collected in the east sector of central Tyrrhenian Sea

Nineteen bacterial isolates were grown in shaken cultures in media containing chitin as carbon source and different additional nitrogen sources such as yeast nitrogen base (YNB), yeast extract (YE), corn steep liquor (CSL) and ammonium sulfate. Strain BM17 showed the highest activity (200 U/l) in medium containing Chitin (1%) and YNB (0.5%). Molecular analysis of the 16S rRNA gene showed that strain BM17 belongs to the species Paenibacillus pabuli (99.72% homology). The enzyme activity started after 12-24 h; exponential enzyme production was recorded from the 24th h and lasted till the 96th h of incubation when activity peaked to decrease thereafter. Medium optimisation was carried out by Response Surface Methodology (RSM) considering the effects of chitin, corn steep liquor and yeast extract. BM17 chitinolytic activity was induced by chitin but the increase of its concentration did not have significant effects on the enzyme activity. By contrast, the nitrogen source, particularly YE, strongly affected the enzyme production.     

Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris

Summary Alcohol oxidase biosynthesis was induced when Pichia pastoris was grown in a medium containing methanol as the sole carbon and energy source. Specific activity was highest during the logarithmic phase of growth (1.22 g acetaldehyde produced/g cell dry wt. per hour), and declined steadily thereafter. The addition of 0.1% (w/v) yeast extract to the methanol growth medium promoted higher biomass production, increased alcohol oxidase specific activity, and contributed to increased enzyme stability under use conditions. When P. pastoris was used for wholecell bioconversions, 30.2 g of ethanol were oxidized to 28 g acetaldehyde in 12 h, at a carbon recovery of 97%. Acetaldehyde concentrations in excess of 1 M were achieved when the concentration of the TRIS buffer, used to chemically trap the acetaldehyde, was increased to 1 M.Issued as NRCC no. 30256Offprint requests to: W. D. Murray     

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.     

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.     

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.

     

Nutrient effects on biocontrol of Penicillium roqueforti by Pichia anomala J121 during airtight storage of wheat

The biocontrol yeast Pichia anomala inhibits the growth of a variety of mold species. We examined the mechanism underlying the inhibition of the grain spoilage mold Penicillium roqueforti by the biocontrol yeast P. anomala J121 during airtight storage. The biocontrol effect in a model grain silo with moist wheat (water activity of 0.96) was enhanced when complex medium, maltose, or glucose was added. Supplementation with additional nitrogen or vitamin sources did not affect the biocontrol activity of the yeast. The addition of complex medium or glucose did not significantly influence the yeast cell numbers in the silos, whether in the presence or absence of P. roqueforti. Mold growth was not influenced by the addition of nutrients, if cultivated without yeast. The products of glucose metabolism, mainly ethanol and ethyl acetate, increased after glucose addition to P. anomala-inoculated treatments. Our results suggest that neither competition for nutrients nor production of a glucose-repressible cell wall lytic enzyme is the main mode of action of biocontrol by P. anomala in this grain system. Instead, the mold-inhibiting effect probably is due to the antifungal action of metabolites, most likely a combination of ethyl acetate and ethanol, derived from glycolysis. The discovery that sugar amendments enhance the biocontrol effect of P. anomala suggests novel ways of formulating biocontrol yeasts.     

Isolation and characterization ofPenicillium funiculosum mutants with enhanced glucose oxidase production

After the mutagenesis ofPenicillium funiculosum with UV light andN-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5–153% higher (in a medium with glucose) and 4–83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutantP. funiculosum NMU95-132 was chosen for further selection work.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

Growth and production of rifamycin oxidase byCurvularia lunata

Curvularia lunata could neither grow nor produce rifamycin oxidase in synthetic media without peptone and yeast extract. Mycelia grown on complex media were tested for the ability to produce rifamycin oxidase in synthetic media. The optimum concentrations of peptone and yeast extract were in the range of 7.5–10 g/L. Five percent inoculum size was found to be optimum for good growth and enzyme production. Addition of metal ions to the cultivation medium increased the enzyme activity.