SciCombinator

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Concept: Penicillium roqueforti

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Although most eukaryotes reproduce sexually at some moment of their life cycle, as much as a fifth of fungal species were thought to reproduce exclusively asexually. Nevertheless, recent studies have revealed the occurrence of sex in some of these supposedly asexual species. For industrially relevant fungi, for which inoculums are produced by clonal-subcultures since decades, the potentiality for sex is of great interest for strain improvement strategies. Here, we investigated the sexual capability of the fungus Penicillium roqueforti, used as starter for blue cheese production. We present indirect evidence suggesting that recombination could be occurring in this species. The screening of a large sample of strains isolated from diverse substrates throughout the world revealed the existence of individuals of both mating types, even in the very same cheese. The MAT genes, involved in fungal sexual compatibility, appeared to evolve under purifying selection, suggesting that they are still functional. The examination of the recently sequenced genome of the FM 164 cheese strain enabled the identification of the most important genes known to be involved in meiosis, which were found to be highly conserved. Linkage disequilibria were not significant among three of the six marker pairs and 11 out of the 16 possible allelic combinations were found in the dataset. Finally, the detection of signatures of repeat induced point mutations (RIP) in repeated sequences and transposable elements reinforces the conclusion that P. roqueforti underwent more or less recent sex events. In this species of high industrial importance, the induction of a sexual cycle would open the possibility of generating new genotypes that would be extremely useful to diversify cheese products.

Concepts: Gene, Genetics, Evolution, Biology, Eukaryote, Fungus, Asexual reproduction, Penicillium roqueforti

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While the extent and impact of horizontal transfers in prokaryotes are widely acknowledged, their importance to the eukaryotic kingdom is unclear and thought by many to be anecdotal. Here we report multiple recent transfers of a huge genomic island between Penicillium spp. found in the food environment. Sequencing of the two leading filamentous fungi used in cheese making, P. roqueforti and P. camemberti, and comparison with the penicillin producer P. rubens reveals a 575 kb long genomic island in P. roqueforti-called Wallaby-present as identical fragments at non-homologous loci in P. camemberti and P. rubens. Wallaby is detected in Penicillium collections exclusively in strains from food environments. Wallaby encompasses about 250 predicted genes, some of which are probably involved in competition with microorganisms. The occurrence of multiple recent eukaryotic transfers in the food environment provides strong evidence for the importance of this understudied and probably underestimated phenomenon in eukaryotes.

Concepts: Archaea, Bacteria, Organism, Eukaryote, Fungus, Animal, Penicillium, Penicillium roqueforti

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Artificial two-dimensional biological habitats were prepared from porous polymer layers and inoculated with the fungus Penicillium roqueforti to provide a living material. Such composites of classical industrial ingredients and living microorganisms can provide a novel form of functional or smart materials with capability for evolutionary adaptation. This allows realization of most complex responses to environmental stimuli. As a conceptual design, we prepared a material surface with self-cleaning capability when subjected to standardized food spill. Fungal growth and reproduction were observed in between two specifically adapted polymer layers. Gas exchange for breathing and transport of nutrient through a nano-porous top layer allowed selective intake of food whilst limiting the microorganism to dwell exclusively in between a confined, well-enclosed area of the material. We demonstrated a design of such living materials and showed both active (eating) and waiting (dormant, hibernation) states with additional recovery for reinitiation of a new active state by observing the metabolic activity over two full nutrition cycles of the living material (active, hibernation, reactivation). This novel class of living materials can be expected to provide nonclassical solutions in consumer goods such as packaging, indoor surfaces, and in biotechnology.

Concepts: Natural selection, Bacteria, Evolution, Metabolism, Nutrition, Organism, Fungus, Penicillium roqueforti

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Fungi exhibit substantial morphological and genetic diversity, often associated with cryptic species differing in ecological niches. Penicillium roqueforti is used as a starter culture for blue-veined cheeses, being responsible for their flavor and color, but is also a common spoilage organism in various foods. Different types of blue-veined cheeses are manufactured and consumed worldwide, displaying specific organoleptic properties. These features may be due to the different manufacturing methods and/or to the specific P. roqueforti strains used. Substantial morphological diversity exists within P. roqueforti and, although not taxonomically valid, several technological names have been used for strains on different cheeses (e.g., P. gorgonzolae, P. stilton). A worldwide P. roqueforti collection from 120 individual blue-veined cheeses and 21 other substrates was analyzed here to determine (i) whether P. roqueforti is a complex of cryptic species, by applying the Genealogical Concordance Phylogenetic Species Recognition criterion (GC-PSR), (ii) whether the population structure assessed using microsatellite markers correspond to blue cheese types, and (iii) whether the genetic clusters display different morphologies. GC-PSR multi-locus sequence analyses showed no evidence of cryptic species. The population structure analysis using microsatellites revealed the existence of highly differentiated populations, corresponding to blue cheese types and with contrasted morphologies. This suggests that the population structure has been shaped by different cheese-making processes or that different populations were recruited for different cheese types. Cheese-making fungi thus constitute good models for studying fungal diversification under recent selection.

Concepts: Evolution, Biology, Species, Fungus, Ascomycota, Penicillium, Cheese, Penicillium roqueforti

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Complex bouquet: A recently developed method for trace analyses combining the advantages of GC-MS and (13) C NMR spectroscopy was applied to investigate the volatiles of Penicillium roqueforti. Besides the main compound, aristolochene, several side products of aristolochene synthase and downstream oxidation products en route to PR toxin were identified, giving insight into the biosynthetic pathway.

Concepts: Scientific method, Amino acid, Adenosine triphosphate, Fungus, Vitamin, Protein biosynthesis, Penicillium roqueforti, Aristolochene

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The Penicillium genera, encompassing about 225 different species of fungi, are naturally present in the environment. These genera are poorly linked to human disease, except for Penicillium marneffei causing septicemia in immunocompromised hosts. Thus, Penicillium species recovered from respiratory tract samples are often considered as inhaled contaminants in the clinical laboratory. However, we report here a case of fungal maxillary sinusitis due to Penicillium roqueforti diagnosed in a 40-year-old female, a teacher, complaining of moderate pain for months in the maxillary sinus and chronic posterior rhinorrhea. CT scanner and MRI enabled a preliminary diagnosis of left maxillary fungus ball-type sinusitis with calcified material seen on CT and marked very low signal in T2 weighted images seen on MRI. Anatomopathological and mycological examination of sinusal content showed septate hyphae. Direct sequencing of the sinusal content revealed P. roqueforti. P. roqueforti has been traditionally used in France for more than 200 years for cheese ripening. However, to our knowledge, this ascomycetous fungus has very rarely been associated in the literature with human disease. P. roqueforti is associated only with cheese worker’s lung, a hypersensitivity pneumonitis affecting employees in blue cheese factories. Other species in the Penicillium genus are reported to cause various disorders such as invasive infection, superficial infection or allergic diseases. P. roqueforti has never previously been reported as a cause of human infection. Thus, we report the first case of fungus ball due to P. roqueforti in an immunocompetent patient.

Concepts: Disease, Fungus, Sinusitis, Allergy, Maxillary sinus, Ascomycota, Penicillium, Penicillium roqueforti

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Moulds food infestation is a heavy dangerous problem for human health and also could generate heavy economic losses. The intelligent packaging using eco-friendly biodegradable biofilm incorporating bioactive natural safe compounds represents a new frontier. This manuscript reports the inhibitory activity of 12 bacterial, fungal and plant metabolites against Penicillium roqueforti and Aspergillus niger. Among them α-costic acid and ungeremine (3 and 12) are the most promising as potential biofungicide against both fungal strains. They inhibited fungal growth by more than 60% respect to the control at 72 h and this activity persisted also at 96 h. Ungeremine showed MIC90 lower than 0.003 mg/mL after 48 h of incubation and of 0.025 mg/mL at 72 h against P. roqueforti. The MIC90 value for A. niger was 0.2 mg/mL at 48 h for both compounds. The α-costic acid showed generally MIC values at 48 and 72 h higher than ungeremine.

Concepts: Bacteria, Eukaryote, Fungus, Mycotoxin, Aspergillus, Ascomycota, Penicillium, Penicillium roqueforti

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The blue cheese-making fungus Penicillium roqueforti produces isofumigaclavine A as the main ergot alkaloid. Recently, genome mining revealed the presence of two DNA loci bearing the genetic potential for its biosynthesis. In this study, a short-chain dehydrogenase/reductase (SDR) from one of the loci was proved to be responsible for the conversion of chanoclavine-I to its aldehyde. Furthermore, a putative gene coding for an enzyme with high homology to Old Yellow Enzymes (OYEs) involved in the ergot alkaloid biosynthesis was found outside the two clusters. Biochemical characterisation of this enzyme, named FgaOx3Pr3, showed that it can indeed catalyse the formation of festuclavine in the presence of a festuclavine synthase FgaFS, as had been observed for other OYEs in ergot alkaloid biosynthesis. Differing from other homologues, FgaOx3Pr3 does not convert chanoclavine-I aldehyde to its shunt products in the absence of FgaFS. Instead, it increases significantly the product yields of several SDRs for the conversion of chanoclavine-I to its aldehyde. Kinetic studies proved that overcoming the product inhibition is responsible for the observed enhancement. To the best of our knowledge, this is the first report on the bifunctionality of an OYE and its synergistic effect with SDRs.

Concepts: DNA, Gene, Genetics, Enzyme, Fungus, Ascomycota, Alkaloid, Penicillium roqueforti

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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.

Concepts: Protein, Bacteria, Amino acid, Enzyme, Fungus, Penicillium, Alkaloid, Penicillium roqueforti

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Aiming at identifying antifungal compounds from plant matrices to be used as ingredients in the bakery industry, a water/salt-soluble extract (WSE) was produced from a legume enzyme hydrolysate, consisting of a mixture of pea, lentil, and faba bean flours, and assayed towards Penicillium roqueforti DPPMAF1. Agar diffusion assays allowed the selection of the optimal processing conditions for hydrolysis. As shown by hyphal radial growth rate, the inhibition was observed towards several fungi, including Aspergillus parasiticus CBS971.97, Penicillium carneum CBS 112297, Penicillium paneum CBS 101032, Penicillium polonicum 112490. A multi-step purification was carried out to identify the active compounds. The antifungal activity was attributed to native proteins (nsLTP, ubiquitin, lectin alpha-1 chain, wound-induced basic protein, defensin-1, defensin-2) and a mixture of peptides, which were released during hydrolysis. Nine peptides were purified and identified as sequences encrypted in legume vicilins, lectins and chitinases. WSE was used as ingredient for making bread under pilot plant conditions. Chemical, structural and sensory characterization of bread showed the lack of significant changes compared to control. The bread made with the legume hydrolysate had a longer shelf-life than that of the control.

Concepts: Protein, Protein structure, Fungus, Yeast, Wheat, Starch, Vicia, Penicillium roqueforti