Optimization of the Medium and Cultivation Conditions of Penicillium roquefortii f39 Producing the Diketopiperazine Alkaloid Roquefortine

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5–2-fold. An increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and the amount of the inoculum. The use of sucrose in the inoculum medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.     

Optimization of the medium and cultivation conditions of Penicillium roquefortii f39 producing the diketopiperazine alkaloid roquefortine

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5-2-fold. The increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and amount of the inoculum. The introduction of sucrose into the growth medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.     

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

Factors Contributing to Roquefortine Yield Variability during Cultivation of Penicillium roquefortii

Variability in the roquefortine yield was shown to be associated with its consumption by the mycelium during isolation of the end product, which depended on temperature, time of culture liquid storage, and biomass concentration. This was also related to the presence in chloroform of chlorocarbonic acid ethyl ester that reacted with roquefortine.     

Regulation aspects of roquefortine production by free and Ca-alginate immobilized mycelia of Penicillium roqueforti

Summary Roquefortine synthesis with free and Ca-alginate immobilized Penicillium roqueforti cells was investigated under different culture conditions. Decreasing Ca-alginate concentration was related to increasing roquefortine production; free cells gave the best results. Formation of roquefortine was three times higher with mannitol and succinate than with sucrose as the carbon source; phosphate inhibited its biosynthesis in free cells by 23% to 32%. Relationships between cell density, ¹⁴C-tryptophan content of cells and roquefortine synthesis were shown. The special morphology of immobilized mycelia was demonstrated.     

Penitrem A and Roquefortine Production by Penicillium commune

Extracts of Penicillium commune, a fungus isolated from cottonseed, showed biological activity in day-old cockerels. Two neurotoxic metabolites were isolated and identified as penitrem A and roquefortine. This is the first report of roquefortine being produced by a fungus other than Penicillium roqueforti as well as the first report of penitrem A and roquefortine being produced in the same culture. Production of these toxins on liquid media and cottonseed was determined.     

Classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites

Strains of available terverticillate penicillium species and varieties were analyzed for profiles of known mycotoxins and other secondary metabolites produced on Czapek yeast autolysate agar (intracellular metabolites) and yeast extract-sucrose agar (extracellular metabolites) by using simple thin-layer chromatography screening techniques. These strains (2,473 in all) could be classified into 29 groups based on profiles of secondary metabolites. Most of these profiles of secondary metabolites were distinct, containing several biosynthetically different mycotoxins and unknown metabolites characterized by distinct colors and retardation factors on thin-layer chromatography plates. Some species (P. italicum and P. atramentosum) only produced one or two metabolites by the simple screening methods. The 29 groups based on profiles of secondary metabolites were known species or subgroups thereof. These species and subgroups were independently identifiable by using morphological and physiological criteria. The species accepted, the number of isolates in each species investigated, and the mycotoxins they produced were: P. atramentosum, 4; P. aurantiogriseum, 510 (group I: penicillic acid and S-toxin and group II: penicillic acid, penitrem A [low frequency], terrestric acid [low frequency], viomellein, and xanthomegnin); P. brevicompactum, 81 (brevianamid A and mycophenolic acid); P. camembertii group I, 38, and group II, 114 (cyclopiazonic acid); P. chrysogenum, 87 (penicillin, roquefortine C, and PR-toxin); P. claviforme, 4 (patulin and roquefortine C); P. clavigerum, 4 (penitrem A); P. concentricum group I, 10 (griseofulvin and roquefortine C), and group II, 3 (patulin and roquefortine C); P. crustosum, 123 (penitrem A, roquefortine C, and terrestric acid); P. echinulatum, 13; P. expansum, 91 (citrinin, patulin, and roquefortine C); P. granulatum, 6 (patulin, penitrem A, and roquefortine C [traces]); P. griseofulvum, 21 (cyclopiazonic acid, griseofulvin, patulin, and roquefortine C); P. hirsutum, 100 (group I: terrestric acid; group II: citrinin, penicillic acid , roquefortine C, and terrestric acid; and group III: roquefortine C and terrestric acid), P. hirsutum group IV, 2 (chaetoglobosin C); P. isariiforme, 1; P. italicum, 41; P. mali, 104; P. roquefortii, 78 (group I: mycophenolic acid, PR-toxin, and roquefortine C and group II: mycophenolic acid, patulin, penicillic acid [low frequency], and roquefortine C); P. viridicatum group I, 634 (brevianamid A [low frequency], penicillic acid, viomellein, and xanthomegnin), P. viridicatum group II and III, 494 (citrinin and ochratoxin A), P. viridicatum group IV, 12 (griseofulvin and viridicatumtoxin). It is proposed that profiles of secondary metabolites be strongly emphasized in any future revision of the penicillia.     

Formation of alkaloids from Penicillium species fungi during growth on wheat kernels

The ability to produce alkaloids has been studied in 13 strains belonging to 10 species of the genus Penicillium. Most of these strains produce identical ranges of alkaloids when grown on wheat grain and synthetic Abe medium. They are roquefortine, 3,12-dihydroroquefortine, and glandicolines A and B in strain P. chrysogenum VKM F-1987; fumigaclavines A and B, festuclavine, and pyroclasine in P. commune VKM F-308, F-3491, and KBP4; agroclavine 1 and epoxyagroclavine 1 in P. fellutanum VKM F-1073; fellutanine A in P. fellutanum F-3020; roquefortine, 3,12-dihydroroquefortine, meleagrin, and glandicolines A and B in P. glandicola VKM F-743; aurantioclavine in P. nalgiovense VKM F-229; isofumigaclavines A and B, festuclavine, roquefortine, and 3,12-dihydroroquefortine in P. roquefortii VKM F-2389; roquefortine, 3,12-dihydroroquefortine, and meleagrin in P. vitale VKM F-3624; roquefortine and oxaline in P. vulpinum VKM F-256; and alpha-cyclopiazonic acid and rugulovasine B in P. viridicatum C-47. No alkaloids were found in P. rugulosum VKM F-352 grown on wheat grain. A simple method is proposed for isolating alkaloids from affected grains.     

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.     

Penicillium aurantiogriseum Dierckx 1901: Producer of Diketopiperazine Alkaloids (Roquefortine and 3,12-Dihydroroquefortine), Isolated from Permafrost

Secondary metabolites of three strains of Penicillium aurantiogriseumisolated from permafrost sediments were identified. It was found that these fungi synthesized the diketopiperazine alkaloids roquefortine and 3,12-dihydroroquefortine. The strain VKM FW-766 synthesized alkaloids in the course of certain growth-related processes. When the strain was grown on a mineral medium, the time courses of the roquefortine and 3,12-dihydroroquefortine concentrations were characterized by biphasic curves.     

Novel Key Metabolites Reveal Further Branching of the Roquefortine/Meleagrin Biosynthetic Pathway

Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.     

Isolation of Penicillium DIERCKX 1901--a producer of diketopiperazine alkaloids rokefortin and 3,12-dihydrokefortin--from permafrost

Secondary metabolites of the three strains of Penicillium aurantiogriseum, isolated from permafrost sediments, were identified. It was found that these fungi synthesized diketopiperazine alkaloids roquefortine and 3,12-dihydroroquefortine. The strain VKM FW-766 synthesized alkaloids in in the course of certain growth-related processes. When the strain was grown on a mineral medium, time courses of roquefortine and 3,12-dihydroroquefortine concentrations were characterized by biphasic curves.     

Antimicrobial action of roquefortine

Summary The effect of roquefortine, a secondary metabolite of Penicillium roqueforti on microorganisms, was investigated. The growth of gram-positive organisms containing hemins was inhibited, whereas Lactobacteria and Clostridia which lack these hemins were only impaired. The growth of gram-negative cells was not affected; therefore, it is supposed that there is no transport of roquefortine into these cells.     

The ciliated protozoa Tetrahymena thermophila as a bionsensor to detect mycotoxins

The cytotoxic effect of four mycotoxins (patulin, diacetoxyscirpenol, roquefortine and T-2 toxin) has been tested on the ciliate Tetrahymena thermophila. This ciliate has been shown to be a very sensitive biosensor to patulin, diacetoxyscirpenol and T-2 toxin. With respect to the roquefortine, this is the first reported bioassay using eukaryotic cells, and results show a lower sensitivity than tests using bacteria. Results are compared with those obtained using other biosensors.     

Silencing of a second dimethylallyltryptophan synthase of <Emphasis Type="Italic">Penicillium roqueforti</Emphasis> reveals a novel clavine alkaloid gene cluster

Penicillium roqueforti produces several prenylated indole alkaloids, including roquefortine C and clavine alkaloids. The first step in the biosynthesis of roquefortine C is the prenylation of tryptophan-derived dipeptides by a dimethylallyltryptophan synthase, specific for roquefortine biosynthesis (roquefortine prenyltransferase). A second dimethylallyltryptophan synthase, DmaW2, different from the roquefortine prenyltransferase, has been studied in this article. Silencing the gene encoding this second dimethylallyltryptophan synthase, dmaW2, proved that inactivation of this gene does not prevent the production of roquefortine C, but suppresses the formation of other indole alkaloids. Mass spectrometry studies have identified these compounds as isofumigaclavine A, the pathway final product and prenylated intermediates. The silencing does not affect the production of mycophenolic acid and andrastin A. A bioinformatic study of the genome of P. roqueforti revealed that DmaW2 (renamed IfgA) is a prenyltransferase involved in isofumigaclavine A biosynthesis encoded by a gene located in a six genes cluster (cluster A). A second three genes cluster (cluster B) encodes the so-called yellow enzyme and enzymes for the late steps for the conversion of festuclavine to isofumigaclavine A. The yellow enzyme contains a tyrosine-181 at its active center, as occurs in Neosartorya fumigata, but in contrast to the Clavicipitaceae fungi. A complete isofumigaclavines A and B biosynthetic pathway is proposed based on the finding of these studies on the biosynthesis of clavine alkaloids.     

Liquid chromatography method for the quantitation of the breast cancer resistance protein ABCG2 inhibitor fumitremorgin C and its chemical analogues in mouse plasma and tissues

Fumitremorgin C (FTC) was recently discovered to be a potent and selective inhibitor of the breast cancer resistance protein (BCRP/ABCG2). FTC was shown to reverse multidrug resistance mediated by BCRP and to increase the cytotoxicity of several anticancer agents in vitro. To support in vivo studies a reverse phase HPLC method with ultraviolet detection was developed to quantitate FTC in mouse plasma and tissues. Further, assay method validation was performed for the determination of FTC in mouse plasma. Plasma standard curves ranged from 0.03 to 30 microg/ml, while the various tissue assay ranges differed to some extent. The sample preparation consisted of acetonitrile precipitation with separation accomplished with a C18 Novapak column and a C18 pre-column utilizing an isocratic mobile phase of ammonium acetate and acetonitrile. UV detection was set at 225 nm for FTC and at 312 nm for roquefortine, the internal standard. The retention times were approximately 9.5 min for FTC and 13.0 min for roquefortine. The recoveries for FTC and roquefortine from plasma were 90.8+/-5.8% and 111.6+/-13.6, respectively. The reported assay can be used for future study of BCRP resistance in vivo in different biological matrices. Further, we found that a more potent analogue of FTC, Ko143, was able to be extracted and detected, with a maximal UV absorbance at 320 nm under the conditions reported.     

Intracellular and extracellular alkaloids of Penicillium roqueforti

The alkaloid composition of mycelium and culture liquid filtrate of the fungus Penicillium roqueforti IBPM-F-141 was studied. The new metabolite--3,12-dihydroroquefortine, a derivative of roquefortine, the main component of the alkaloid fraction of this culture, has been isolated for the first time. The structure of 3,12-dihydroroquefortine was determined by chemical and physico-chemical methods. In addition to roquefortine and 3,12-dihydroroquefortine, representatives of a new alkaloid group, the clavine alkaloids, e. g. isofumigaclavine A, isofumigaclavine B and festuclavine, were also isolated and identified. The data on the content of these compounds in mycelium and culture medium are presented.     

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     

Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Production of Alkaloids by Fungi of the Genus PenicilliumGrown on Wheat Grain

The ability to produce alkaloids has been studied in 13 strains belonging to ten species of the genus Penicillium. Most of these strains produce identical ranges of alkaloids when grown on wheat grain and synthetic Abe's medium. These are roquefortine, 3,12-dihydroroquefortine, and glandicolines A and B in strain P. chrysogenum VKM F-1987; fumigaclavines A and B, festuclavine, and pyroclavine in P. commune VKM F-308, F-3491, and KBP4; agroclavine 1 and epoxyagroclavine 1 in P. fellutanum VKM F-1073; fellutanine A in P. fellutanum F-3020; roquefortine, 3,12-dihydroroquefortine, meleagrin, and glandicolines A and B in P. glandicola VKM F-743; aurantioclavine in P. nalgiovense VKM F-229; isofumigaclavines A and B, festuclavine, roquefortine, and 3,12-dihydroroquefortine in P. roquefortii VKM F-2389; roquefortine, 3,12-dihydroroquefortine, and meleagrin in P. vitale VKM F-3624; roquefortine and oxaline in P. vulpinum VKM F-256; and -cyclopiazonic acid and rugulovasine B in P. viridicatum C-47. No alkaloids were found in P. rugulosum VKM F-352 grown on wheat grain. A simple method is proposed for isolating alkaloids from affected grain.