Elaborated regulation of griseofulvin biosynthesis in Penicillium griseofulvum and its role on conidiation and virulence.

Penicilium griseofulvum, the causal agent of apple blue mold, is able to produce in vitro and on apple a broad spectrum of secondary metabolites (SM), including patulin, roquefortine C and griseofulvin. Among them, griseofulvin is known for its antifungal and antiproliferative activity, and has received interest in many sectors, from medicine to agriculture. The biosynthesis of SM is finely regulated by filamentous fungi and can involve global regulators and pathway specific regulators, which are usually encoded by genes present in the same gene cluster as the backbone gene and tailoring enzymes. In the griseofulvin gene cluster, two putative transcription factors were previously identified, encoded by genes gsfR1 and gsfR2, and their role has been investigated in the present work. Analysis of P. griseofulvum knockout mutants lacking either gene suggest that gsfR2 forms part of a different pathway and gsfR1 exhibits many spectra of action, acting as regulator of griseofulvin and patulin biosynthesis and influencing conidia production and virulence on apple. The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen. The influence of carbon and nitrogen on griseofulvin production was further investigated and verified, revealing a complex network of response and confirming the central role of gsfR1 in many processes in P. griseofulvum.

Complexity generation in fungal polyketide biosynthesis: a spirocycle-forming P450 in the concise pathway to the antifungal drug griseofulvin.

Griseofulvin (1) is a spirocyclic fungal natural product used in treatment of fungal dermatophytes. Formation of the spirocycle, or the grisan scaffold, from a benzophenone precursor is critical for the activity of 1. In this study, we have systematically characterized each of the biosynthetic enzymes related to the biogenesis of 1, including the characterization of a new polyketide synthase GsfA that synthesizes the benzophenone precursor and a cytochrome P450 GsfF that performs oxidative coupling between the orcinol and the phloroglucinol rings to yield the grisan structure. Notably, the finding of GsfF is in sharp contrast to the copper-dependent dihydrogeodin oxidase that performs a similar reaction in the geodin biosynthetic pathway. The biosynthetic knowledge enabled the in vitro total biosynthesis of 1 from malonyl-CoA using all purified enzyme components. This work therefore completely maps out the previously unresolved enzymology of the biosynthesis of a therapeutically relevant natural product.

Identification of the viridicatumtoxin and griseofulvin gene clusters from Penicillium aethiopicum.

Penicillium aethiopicum produces two structurally interesting and biologically active polyketides: the tetracycline-like viridicatumtoxin 1 and the classic antifungal agent griseofulvin 2. Here, we report the concurrent discovery of the two corresponding biosynthetic gene clusters (vrt and gsf) by 454 shotgun sequencing. Gene deletions confirmed that two nonreducing PKSs (NRPKSs), vrtA and gsfA, are required for the biosynthesis of 1 and 2, respectively. Both PKSs share similar domain architectures and lack a C-terminal thioesterase domain. We identified gsfI as the chlorinase involved in the biosynthesis of 2, because deletion of gsfI resulted in the accumulation of decholorogriseofulvin 3. Comparative analysis with the P. chrysogenum genome revealed that both clusters are embedded within conserved syntenic regions of P. aethiopicum chromosomes. Discovery of the vrt and gsf clusters provided the basis for genetic and biochemical studies of the pathways.

Solid-state fermentation for production of griseofulvin on rice bran using Penicillium griseofulvum.

Griseofulvin is a secondary metabolite produced from fungal species that have morphology suitable for solid-state fermentation (SSF). Reports on production of griseofulvin by SSF are scarce. The present work investigates SSF for griseofulvin production, optimization of its process parameters vis-à-vis the conventional submerged fermentation and its downstream processing from the same. Rice bran adjusted to an initial moisture content (IMC) of 50% (v/w) inoculated with 1 mL of a suspension of 10(6) spores/mL under agitation at 250 rpm containing the modified Czapek-Dox medium and additional 0.1% choline chloride as a precursor gave a yield of griseofulvin in 9 days that was comparable to submerged fermentation after 28 days. The yield of griseofulvin (microg/g dry biomass) was comparable in SSF and submerged fermentation. The biomass was estimated by estimation of chitin. Discussions on the effect of each parameter in SSF have also been included.

Penicillium persicinum, a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai Province, China.

A unique Penicillium isolate from Chinese soil with terverticillate penicilli and ellipsoidal to cylindrical smooth-walled conidia, produces, in addition to the common metabolite ergosterol, copious amounts of an unknown peach-red pigment and the following secondary metabolites: griseofulvin, dechlorogriseofulvin, lichexanthone, roquefortine C, roquefortine D, chrysogine, 2-pyrovoylaminobenzamide, 2-acetyl-quinazolin-4(3H)-one. This isolate, CBS 111235, is described as Penicillium persicinum sp. nov., which belongs to subgenus Penicillium section Chrysogena but is morphologically similar to P. italicum. On the basis of the production of secondary metabolites it resembles P. griseofulvum and P. coprophilum. Sequence data using part of the beta-tubulin gene showed that it is phylogenetically related to P. chrysogenum and P. aethiopicum in section Chrysogena with which it shares both secondary metabolites and ability to grow at 37 degrees C.

Supercritical carbon dioxide extraction of griseofulvin from the solid matrix obtained after solid-state fermentation.

Globally there is an increasing concern to minimize the use of organic solvents, particularly the chlorinated ones because of their suspected human carcinogenicity. The use of ecofriendly carbon dioxide as an alternative to organic solvents would be appropriate in the perspective of green technology. Supercritical carbon dioxide (SC-CO(2)) extraction is suitable for extraction of nonpolar compound with molecular weights less than 400. Griseofulvin is an antifungal antibiotic having a molecular weight of 353, making it amenable to SC-CO(2) extraction. This work brings out the potential of supercritical carbon dioxide extraction (SCFE) for downstream processing of griseofulvin from the solid matrix obtained after solid-state fermentation (SSF). The optimized conditions for SCFE of griseofulvin from dried media after SSF were a flow rate of 0.4 L/min, temperature of 60 degrees C, and contact time of 90 min (30 min static + 60 min dynamic) at a pressure of 450-455 bar.

[Biosynthesis and microbial transformation of griseofulvin and carcinogenesis and prevention of cancer by estrogens].

In the biosynthetic study of griseofulvin by Penicillium urticae and microbial transformation of (-)- and (+)-dehydrogriseofulvin and their derivatives by Streptomyces cinereocrocatus excellent informations were obtained from 2H-NMR spectroscopy. In the reduction of (-)-dehydrogriseofulvin into (+)-griseofulvin by a partially purified enzyme system of S. cinereocrocatus, the origin of the 6' alpha-hydrogen of (+)-griseofulvin was a hydride ion donated by pro-4R-hydrogen of NADPH. In connection with the study of carcinogenesis, diethylstilbestrol (DES) was proved to disrupt microtubules in vitro. The other synthetic estrogens, E,E-dienestrol, meso-hexestrol, and dl-hexestrol were inhibitors of microtubule assembly in vitro, and induced twisted ribbon structures or ribbon-sheet-microtubules from microtubule proteins. Next, the effects of DES and its methyl ethers on the chromosome of and the cellular microtubule architecture, revealed by fluorescent anti-tubulin antibody, of Chinese hamster V79 cells were examined, and further estradiol-17 beta was proved to exhibit higher microtubule-disruptive activity than DES in V79 cells. Furthermore, cytoplasmic microtubules in the human breast cancer cell lines MCF-7 and MDA-MB-231, estrogen receptor-positive and -negative cell lines, respectively, were disrupted equally by estradiol-17 beta. Then, natural estrogens and their derivatives comprising 30 compounds in total were tested in Chinese hamster V79 cells, proving that 2-methoxyestradiol showed the strongest activity (EC50: 2 microM) to disrupt microtubules. Further, in the assay of indenestrol A, a metabolite of DES, indenestrol B and their monomethyl ethers, the 4'-methyl ether of [(-)-3S]-indenestrol B exhibited both the strongest cytotoxicity in, and greatest disruption of the cellular microtubules of V79 cells, and no correlation with the affinity for estrogen receptors was shown.

Differentiation of Penicillium griseofulvum Dierckx isolates by enzyme assays and by patulin and griseofulvin analyses.

The production of patulin and griseofulvin by 49 different isolates of Penicillium griseofulvum Dierckx was analyzed by high-performance liquid chromatography. Eleven isolates were obtained from pistachio nuts, 37 were obtained from wheat seeds, and 1 was obtained from the American Type Culture Collection. Activities of 19 enzymes were also assayed by the API ZYM system. From these results it may be deduced that there are two different groups among the strains tested which cannot be distinguished by morphological and cultural characteristics. One group of isolates did not produce detectable amounts of patulin and griseofulvin when grown in sucrose-yeast extract and Wickerham media, while enzymatic activities were quantitatively distinct from the other group, which produced patulin and griseofulvin in variable proportions. Leucine arylamidase, phosphoamidase, and beta-D-glucosidase are the main enzymes with differing activities between the two groups. Differences in physiological characteristics among isolates of a single species reveal shortcomings in the classification of the penicillia based only on morphological criteria. Thus, determination of the ability to yield mycotoxins and antibiotics as well as determination of enzymatic activities appear to be very valuable tools in the taxonomy of these fungi and for food toxicology.

Production of patulin and griseofulvin by a strain of Penicillium griseofulvum in three different media.

Production of patulin and griseofulvin by a strain of Penicillium griseofulvum is described. Mycelial dry weight, pH and production of patulin and griseofulvin were assayed in a minimal and a complete medium; patulin or griseofulvin production was assayed in apple juice.

A novel process for the production of penitrem mycotoxins by submerged fermentation of Penicillium nigricans.

A strain of Penicillium nigricans, which produces both the antifungal antibiotic griseofulvin and tremorgenic penitrem mycotoxins concurrently in static liquid culture, also elaborated both metabolites in submerged culture when stimulated by calcium chloride to sporulate. Maximum yield of penitrems (60 mg l-1) occurred within 5 d in a 60 l stirred fermenter, thus constituting the first significant process for penitrem production in submerged culture.

[Effect of supplementary carbohydrate and nitrogen sources on metabolite concentration in the mycelium of Penicillium nigricans Thom]. / Vliianie dopolnitel'nykh istochnikov ugleroda i azota na kontsentratsiiu nekotorykh metabolitov v mitselii Penicillium nigricans Thom.

The effect of additional sources of carbon (glucose) and nitrogen (NaNO3) on the dynamics of the levels of glucose-6-phosphate (G-6-P), phosphoenolpyruvate (PEP) and pyruvate in the mycelium of the productive strain of P. nigricans synthesizing griseofulvin was studied. It was found that the PEP level was characterized by stability, while the levels of G-6-P and pyruvate depended on the mycelium age at the time of the source addition, the addition type, i.e. addition of a source alone or a mixture of sources and the way of addition (single or divided).

[Effect of additions of the carbon and nitrogen sources on the NADP+ and NAD+ level in the mycelium of a producing strain of P. nigricans Thom]. / Vliianie dobavok istochnikov ugleroda i azota na uroven' NADF+ i NAD+ v mitselii produktivnogo shtamma P. nigricans Thom.

The levels of NADP+ and NAD+ in the mycelium of a cultured strain of P. nigricans Thom grown on the mineral medium with 2 per cent of glucose were studied as dependent on the time and method of addition of glucose. NaNO3 or their mixture to the medium, i. e. a single addition simultaneously with the spore inoculation or on the 7th day of the culture development, divided additions on the 5th--7th days. It was shown that addition of the above sources simultaneously with inoculation affected the levels of both dinucleotides by the 5th and 8th days. Analogous additions on the 7th day in 24 hours decreased the level of NAD+, while the NADP+ concentration increased after addition of a mixture of glucose and NANO3 and remained unchanged on their separate addition. When the above sources were added in divided doses the level of NADP+ increased till the 7th day and that of NAD+ till the end of the experiment (the 8th day).

[Effect of carbon and nitrogen source additions on the development of a productive strain of P. nigricans Thom and on the level of adenylates in its mycelium]. / Vliianie dobavok istochnikov ugleroda i azota na razvitie produktivnogo shtamma P. nigricans Thom i uroven' adenilatov v ego mitselii.

Growth of the cultured strain og P. nigricans and dynamics of the adenylate levels in its mycelium on mineral media with 2 per cent of glucose were studied in relation to the means and time of addition of glucose, NaNO3 or their mixture to the medium. It was shown that the maximum yield of the mycelium could be obtained with addition of glucose once at the moment of inoculation. The mixture of glucose with NaNO3 provided even higher yields of the biomass but only with its fractional addition. Introduction of additional amounts of NaNO3 at the moment of inoculation and during the growth phase (5 days) inhibited the subsequent development of the culture providing stable levels of ATP and ADP, while introduction of NaNO3 on the 7th day stimulated the culture growth and the antibiotic yield. The use of NaNO3 in the mixture with glucose eliminated inhibition and increased the ratio of ATP to ADP and the antibiotic yield.

[Action of N-nitroso-N-methylbiuret on Penicillium nigricans Thom and the protective effect in UV irradiation]. / Deistvie N-nitroso-N-metilbiureta na Penicillium nigricans Thom i éffekt zashchity pri UF-obluchenii

N-Nitrozo-N-methylbiuret (NMB) in a concentration of 0.2% and exposition for 2,3,4 and 6 hours with the survival rate of the conidia of 4-25% induced slight morphological variation of the griseofulvin-producing organism and increased the variation rate with respect to the antibiotic production by 40-140%. Preliminary treatment of the conidia with NMB decreased the lethal effect of UV-rays approximately by 15-25 times. The protective range with respect to morphological variation was 10 times lower. With an increase in the toxicity of NMB its protective effect in irradiation with UV-light decreased.

Mycotoxin-producing potential of mold flora of dried beans.

To evaluate the potential for mycotoxin production by molds in dried beans, the mold flora of 114 samples was determined both before and after surface disinfection of the beans with 5% NaOCl. Surface disinfection substantially reduced mold incidence, indicating that contamination was mainly on the surface. The flora, both before and after disinfection, was dominated by species of the Aspergillus glaucus group, the toxicogenic species A ochracues, Penicillium cyclopium, and P. viridicatum, and species of Alternaria, Cladosporium, and Fusarium. The toxicogenic species Aspergillus flavis, A. versicolor, Penicillium Citrinum, P. expansum, P. islandicum, and P. urticae were encountered less frequently. Of 209 species of Aspergillus and Penicillium screened for mycotoxin production on sterile rice substrate, 114 produced one or more of the following mycotoxins: A. flavus, aflatoxins; A. ochraceus, ochratoxins; A. nidulans, A. unguis, and A. versicolor, sterigmatocystin; P. cyclopium, penicillic acid; P. citrinum and P. viridicatum, citrinin; P. urticae, patulin and griseofulvin. Sterigmatocystin production by A. unguis is reported for the first time.