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Specific roquefortine C production by the Penicillium roqueforti parental strain CECT 2905 (line) and the transformant PRG42-7 (circles) when cultured in YES medium.
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Heterotrimeric G protein signaling regulates many processes in fungi, such as development, pathogenicity, and secondary metabolite biosynthesis. For example, the Galpha subunit Pga1 from Penicillium chrysogenum regulates conidiation and secondary metabolite production in this fungus. The dominant activating allele, pga1G42R, encoding a constitutive...
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... of the constitutively active Pga1 α α sub- unit in the production of the secondary meta- bolite roquefortine C. In the parental strain, the high- est levels of roquefortine C production occurred between day 16 and day 21 of culture, with a peak of 0.4 μg/mg of dry mycelium at day 18 (Fig. 4). Afterwards, roquefortine C levels quickly fell to initial levels. In the transformant PRG42-7, car- rying the dominant activating pga1 G42R allele, the production of roquefortine C increased, reaching 0.7 μg/mg of dry mycelium at day 18. Moreover, in contrast to the parental strain, the transformant maintained high levels of roquefor- tine C (always >0.5 μg/mg of dry mycelium) after day 18. This result indicates that the constitutively active Pga1 G42R α subunit should stimulate the production of roquefortine C, confirming the importance of G-protein-mediated signaling in regulating the production of this secondary metabolite. In addition to the increase in mycotoxin production, differences in color and aroma of the liquid cultures were ...
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... The extraction and HPLC analyses of roquefortine C, mycophenolic acid, and andrastin A were performed following the protocols described by Torrent et al. [19], Del-Cid et al. [7], and Rojas-Aedo et al. [12], respectively. The production of these specialized metabolites was normalized by the dry weight of fungal mycelia, as described by García-Rico et al. [18]. ...
The regulation of fungal specialized metabolism is a complex process involving various regulators. Among these regulators, LaeA, a methyltransferase protein originally discovered in Aspergillus spp., plays a crucial role. Although the role of LaeA in specialized metabolism has been studied in different fungi, its function in Penicillium roqueforti remains unknown. In this study, we employed CRISPR-Cas9 technology to disrupt the laeA gene in P. roqueforti (PrlaeA) aiming to investigate its impact on the production of the specialized metabolites roquefortine C, mycophenolic acid, and andrastin A, as well as on asexual development, because they are processes that occur in the same temporal stages within the physiology of the fungus. Our results demonstrate a substantial reduction in the production of the three metabolites upon disruption of PrlaeA, suggesting a positive regulatory role of LaeA in their biosynthesis. These findings were further supported by qRT-PCR analysis, which revealed significant downregulation in the expression of genes associated with the biosynthetic gene clusters (BGCs) responsible for producing roquefortine C, mycophenolic acid, and andrastin A in the ΔPrlaeA strains compared with the wild-type P. roqueforti. Regarding asexual development, the disruption of PrlaeA led to a slight decrease in colony growth rate, while conidiation and conidial germination remained unaffected. Taken together, our results suggest that LaeA positively regulates the expression of the analyzed BGCs and the production of their corresponding metabolites in P. roqueforti, but it has little impact on asexual development.
... Heterotrimeric G proteins, particularly α-subunits, are important in fungi and have been involved mainly in the regulation of growth, asexual reproduction, and secondary metabolism [85]. García-Rico et al. [86] isolated and characterized the pga1 gene from P. chrysogenum, and they decided to perform the heterologous expression of a mutant version of this gene in P. roqueforti [87]. For this purpose, they used a plasmid containing a mutant version of pga1 (named pga1 G42R ), which encodes a protein where a glycine is replaced by arginine at Position 42, producing a "constitutively active" Pga1 protein that is always signaling [88]. ...
... The introduction of this "constitutive" allele in P. roqueforti produced several phenotypic effects. In the case of secondary metabolism, García-Rico et al. [87] measured the production of Roquefortine C during 30 days in a strain of P. roqueforti containing the pga1 G42R allele and observed drastic changes in the production of this secondary metabolite as compared with the wild-type strain. Namely, the strain containing pga1 G42R increased production of the mycotoxin, reaching 0.7 µg/mg of dry mycelium at day 18, whereas the wild-type strain produced 0.4 µg/mg of dry mycelium at the same day. ...
... Namely, the strain containing pga1 G42R increased production of the mycotoxin, reaching 0.7 µg/mg of dry mycelium at day 18, whereas the wild-type strain produced 0.4 µg/mg of dry mycelium at the same day. Interestingly, the levels of Roquefortine C were higher in the pga1 G42R strain throughout a culture period ranging between 10-30 days [87]. These results suggested that pga1 has a positive effect on the production of Roquefortine C in P. roqueforti [87]. ...
Filamentous fungi are an important source of natural products. The mold Penicillium roqueforti, which is well-known for being responsible for the characteristic texture, blue-green spots, and aroma of the so-called blue-veined cheeses (French Bleu, Roquefort, Gorgonzola, Stilton, Cabrales, and Valdeón, among others), is able to synthesize different secondary metabolites, including andrastins and mycophenolic acid, as well as several mycotoxins, such as Roquefortines C and D, PR-toxin and eremofortins, Isofumigaclavines A and B, festuclavine, and Annullatins D and F. This review provides a detailed description of the biosynthetic gene clusters and pathways of the main secondary metabolites produced by P. roqueforti, as well as an overview of the regulatory mechanisms controlling secondary metabolism in this filamentous fungus.
... Among the ex-type strains obtained from the neotype IMI 024313, P. roqueforti CECT 2905 T is one of the most widely used. Many molecular studies have been carried out in this ex-type strain, including the analysis of regulatory genes of development and metabolism (García-Rico et al. 2009;Gil-Durán et al. 2015;Torrent et al. 2017;Rojas-Aedo et al. 2018), and importantly, P. roqueforti CECT 2905 T has been used to demonstrate the functionality of all biosynthetic gene clusters (BGCs) for the production of secondary metabolites characterized so far in this fungal species (Hidalgo et al. 2014;Kosalková et al. 2015;Del-Cid et al. 2016;Fernández-Bodega et al. 2017;Rojas-Aedo et al. 2017). ...
Draft genomes of Penicillium roqueforti, Fusarium sororula, Chalaropsis populi, and Chrysoporthe puriensis are presented. Penicillium roqueforti is a model fungus for genetics, physiological and metabolic studies, as well as for biotechnological applications. Fusarium sororula and Chrysoporthe puriensis are important tree pathogens, and Chalaropsis populi is a soil-borne root-pathogen. The genome sequences presented here thus contribute towards a better understanding of both the pathogenicity and biotechnological potential of these species.
... Several aspects of the regulation of roquefortine C biosynthesis have been studied in some detail in Penicillium species. These studies indicate that the production of this compound is regulated by an α-subunit from a heterotrimeric G protein, the conidiation-specific protein BrlA, and Sfk1, a transmembrane protein involved in the phosphoinositide second messengers' pathway (García-Rico et al., 2008;García-Rico et al., 2009;Qin et al., 2013;Torrent et al., 2017). Regarding andrastin A and mycophenolic acid, to date only Sfk1 has been involved in the regulation of their production (Torrent et al., 2017). ...
Penicillium roqueforti is used in the production of several kinds of ripened blue-veined cheeses. In addition, this fungus produces interesting secondary metabolites such as roquefortine C, andrastin A and mycophenolic acid. To date, there is scarce information concerning the regulation of the production of these secondary metabolites. Recently, the gene named pcz1 (Penicillium C6 zinc domain protein 1) was described in P. roqueforti, which encodes for a Zn(II)2Cys6 protein that controls growth and developmental processes in this fungus. However, its effect on secondary metabolism is currently unknown. In this work, we have analyzed how the overexpression and down-regulation of pcz1 affect the production of roquefortine C, andrastin A and mycophenolic acid in P. roqueforti. The three metabolites were drastically reduced in the pcz1 down-regulated strains. However, when pcz1 was overexpressed, only mycophenolic acid was overproduced while, on the contrary, levels of roquefortine C and andrastin A were diminished. Importantly, these results match the expression pattern of key genes involved in the biosynthesis of these metabolites. Taken together, our results suggest that Pcz1 plays a key role in regulating secondary metabolism in the fungus Penicillium roqueforti.
... Thus, this protein negatively affects growth, sporulation, and thermal and osmotic stress resistance in P. roqueforti (García-Rico et al., 2007, 2008a. Furthermore, Gαi stimulates germination and increases the production of the secondary metabolite roquefortine C (García-Rico et al., 2009). ...
... Roquefortine C production was normalized by dry weight of fungal mycelia. For this purpose, the mycelium from each sample was dried as described by García-Rico et al. (2009). ...
... Spore germination is triggered by environmental factors, which are sensed and transduced by signal transduction pathways that have been studied in detail in few species (Fillinger et al., 2002;Doehlemann et al., 2006;Krijgsheld et al., 2013). As in other fungi, P. roqueforti Gαi signaling has been demonstrated to control conidial germination (García-Rico et al., 2009) and recently it was suggested that pcz1 may be participating in this Gαi-signaling pathway controlling conidial germination (Gil-Durán et al., 2015). Here we show for the first time that sfk1 is also involved in the regulation of conidial germination. ...
The sfk1 (suppressor of four kinase) gene has been mainly studied in Saccharomyces cerevisiae, where it was shown to be involved in growth and thermal stress resistance. This gene is widely conserved within the phylum Ascomycota. Despite this, to date sfk1 has not been studied in any filamentous fungus. Previously, we found that the orthologous of sfk1 was differentially expressed in a strain of Penicillium roqueforti with an altered phenotype. In this work, we have performed a functional characterization of this gene by using RNAi-silencing technology. The silencing of sfk1 in P. roqueforti resulted in decreased apical growth and the promotion of conidial germination, but interesting, it had no effect on conidiation. In addition, the attenuation of the sfk1 expression sensitized the fungus to osmotic stress, but not to thermal stress. RNA-mediated gene-silencing of sfk1 also affected cell wall integrity in the fungus. Finally, the silencing of sfk1 depleted the production of the main secondary metabolites of P. roqueforti, namely roquefortine C, andrastin A, and mycophenolic acid. To the best of our knowledge this is the first study of the sfk1 gene in filamentous fungi.
... Fungal Ga subunits have been classified into three subgroups (I, II, and III) (B€ olker 1998;Li et al. 2007). Ga subunits from subgroup I (hereafter Gai) are implicated in regulating several biological processes such as conidiation (Yu et al. 1996;Ivey et al. 2002;Garc ıa-Rico et al. 2008a), conidial germination (Truesdell et al. 2000;Eaton et al. 2012), vegetative growth (Liu & Dean 1997;Yang & Borkovich 1999), stress resistance (Garc ıa- Rico et al. 2009;Garc ıa-Rico et al. 2011), and the production of proteases (Emri et al. 2008;Tan et al. 2009) and secondary metabolites (Calvo et al. 2002;Yu & Keller 2005;Garc ıa-Rico et al. 2009), among others. ...
... Fungal Ga subunits have been classified into three subgroups (I, II, and III) (B€ olker 1998;Li et al. 2007). Ga subunits from subgroup I (hereafter Gai) are implicated in regulating several biological processes such as conidiation (Yu et al. 1996;Ivey et al. 2002;Garc ıa-Rico et al. 2008a), conidial germination (Truesdell et al. 2000;Eaton et al. 2012), vegetative growth (Liu & Dean 1997;Yang & Borkovich 1999), stress resistance (Garc ıa- Rico et al. 2009;Garc ıa-Rico et al. 2011), and the production of proteases (Emri et al. 2008;Tan et al. 2009) and secondary metabolites (Calvo et al. 2002;Yu & Keller 2005;Garc ıa-Rico et al. 2009), among others. ...
... While it has been described that the disruption of Gai affects conidial germination in some fungi (Truesdell et al. 2000;Eaton et al. 2012), in other cases Gai does not seem to be involved in this process (Jain et al. 2002;Mukherjee et al. 2004). Further, in the fungus Penicillum roqueforti and Penicillum chrysogenum Gai stimulates germination in the absence of any carbon source (Garc ıa- Rico et al. 2009;Garc ıa-Rico et al. 2011). ...
The fungus Penicillium camemberti is widely used in the ripening of various bloomy-rind cheeses. Several properties of P. camemberti are important in cheese ripening, including conidiation, growth and enzyme production, among others. However, the production of mycotoxins such as cyclopiazonic acid during the ripening process by P. camemberti has raised concerns among consumers that demand food with minimal contamination. Here we show that overexpressing an α-subunit from the subgroup I of the heterotrimeric G protein (Gαi) influences several of these processes: it negatively affects growth in a media-dependent manner, triggers conidial germination, reduces the rate of sporulation, affects thermal and osmotic stress resistance, and also extracellular protease and cyclopiazonic acid production. Our results contribute to understanding the biological determinants underlying these biological processes in the economically important fungus Penicillium camemberti.
... The quantity of andrastin A obtained was normalized to the dry weight of the fungal mycelia. For this purpose, the mycelium of each P. roqueforti strain was dried as described before (García-Rico et al., 2009). ...
Penicillium roqueforti is a filamentous fungus involved in the ripening of several kinds of blue cheeses. In addition, this fungus produces several secondary metabolites, including the meroterpenoid compound andrastin A, a promising antitumoral compound. However, to date the genomic cluster responsible for the biosynthesis of this compound in P. roqueforti has not been described. In this work, we have sequenced and annotated a genomic region of approximately 29.4 kbp (named the adr gene cluster) that is involved in the biosynthesis of andrastin A in P. roqueforti. This region contains ten genes, named adrA, adrC, adrD, adrE, adrF, adrG, adrH, adrI, adrJ and adrK. Interestingly, the adrB gene previously found in the adr cluster from P. chrysogenum, was found as a residual pseudogene in the adr cluster from P. roqueforti. RNA-mediated gene silencing of each of the ten genes resulted in significant reductions in andrastin A production, confirming that all of them are involved in the biosynthesis of this compound. Of particular interest was the adrC gene, encoding for a major facilitator superfamily transporter. According to our results, this gene is required for the production of andrastin A but does not have any role in its secretion to the extracellular medium. The identification of the adr cluster in P. roqueforti will be important to understand the molecular basis of the production of andrastin A, and for the obtainment of strains of P. roqueforti overproducing andrastin A that might be of interest for the cheese industry.
... The quantity of andrastin A obtained was normalized to the dry weight of the fungal mycelia. For this purpose, the mycelium of each P. roqueforti strain was dried as described before (García-Rico et al., 2009). ...
Penicillium roqueforti is a filamentous fungus involved in the ripening of several kinds of blue cheeses. In addition, this fungus produces several secondary metabolites, including the meroterpenoid compound andrastin A, a promising antitumoral compound. However, to date the genomic cluster responsible for the biosynthesis of this compound in P. roqueforti has not been described. In this work, we have sequenced and annotated a genomic region of approximately 29.4 kbp (named the adr gene cluster) that is involved in the biosynthesis of andrastin A in P. roqueforti. This region contains ten genes, named adrA, adrC, adrD, adrE, adrF, adrG, adrH, adrI, adrJ and adrK. Interestingly, the adrB gene previously found in the adr cluster from P. chrysogenum, was found as a residual pseudogene in the adr cluster from P. roqueforti. RNA-mediated gene silencing of each of the ten genes resulted in significant reductions in andrastin A production, confirming that all of them are involved in the biosynthesis of this compound. Of particular interest was the adrC gene, encoding for a major facilitator superfamily transporter. According to our results, this gene is required for the production of andrastin A but does not have any role in its secretion to the extracellular medium. The identification of the adr cluster in P. roqueforti will be important to understand the molecular basis of the production of andrastin A, and for the obtainment of strains of P. roqueforti overproducing andrastin A that might be of interest for the cheese industry.
... En estas dos especies, los resultados indican que G␣I desempeña un papel relevante en las etapas tempranas de la germinación. Posteriormente, nuestro grupo de trabajo describió que la activación constitutiva de la señal mediada por la subunidad G␣I de P. roqueforti desencadena la germinación aun en un entorno sin fuentes de carbono, lo que sugiere además que dicho efecto está relacionado con el incremento en los niveles de AMP cíclico intracelular 21 . Al examinar con mayor profundidad el proceso en Penicillium chrysogenum se observó que la deleción de esta subunidad ralentizó la cinética de germinación y disminuyó la tasa de conidias germinadas, un efecto no tan drástico como el documentado en C. trifolii, mientras que la activación constitutiva de la señal produjo resultados similares a los observados en P. roqueforti 21,24 . ...
... Posteriormente, nuestro grupo de trabajo describió que la activación constitutiva de la señal mediada por la subunidad G␣I de P. roqueforti desencadena la germinación aun en un entorno sin fuentes de carbono, lo que sugiere además que dicho efecto está relacionado con el incremento en los niveles de AMP cíclico intracelular 21 . Al examinar con mayor profundidad el proceso en Penicillium chrysogenum se observó que la deleción de esta subunidad ralentizó la cinética de germinación y disminuyó la tasa de conidias germinadas, un efecto no tan drástico como el documentado en C. trifolii, mientras que la activación constitutiva de la señal produjo resultados similares a los observados en P. roqueforti 21,24 . Los resultados también sugieren la participación del AMP cíclico en la inducción de la germinación en P. chrysogenum 24 . ...
... Al realizar un estudio similar en Aspergillus fumigatus se encontró que gpaA (que codifica la subunidad G␣I) es uno de los genes que se transcriben casi de manera constitutiva en la fase de dormancia de la conidia, y su expresión se incrementa alrededor de 7 veces después de 30 min de inducida la germinación 46 . Estos resultados demuestran la participación de G␣I en las fases tempranas de la germinación en A. niger y A. fumigatus, y son consistentes con lo observado en P. chrysogenum, P. roqueforti, C. heterostrophus y C. trifolii 21,24,37,86 . Esta participación preponderante de G␣I en la germinación conidial en A. niger y A. fumigatus contradice lo observado en A. nidulans, donde el rol central es desempeñado por la subunidad G␣III 45 . ...
The phylum Ascomycota comprises about 75% of all the fungal species described, and includes species of medical, phytosanitary, agricultural, and biotechnological importance. The ability to spread, explore, and colonise new substrates is a feature of critical importance for this group of organisms. In this regard, basic processes such as conidial germination, the extension of hyphae and sporulation, make up the backbone of development in most filamentous fungi. These processes require specialised morphogenic machinery, coordinated and regulated by mechanisms that are still being elucidated. In recent years, substantial progress has been made in understanding the role of the signalling pathway mediated by heterotrimericG proteins in basic biological processes of many filamentous fungi. This review focuses on the role of the alpha subunits of heterotrimericG proteins in the morphogenic processes of filamentous Ascomycota.
Copyright © 2016 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.
... In several fungal genomes, G-protein coupled receptors with putative nitrogen-sensing function have been identified. Subsequent G-protein mediated signaling not only triggers the production of suitable catabolic enzymes, but also the production of secondary metabolites (Garcia-Rico et al. 2009;Li et al. 2007;Marzluf 1997). ...
... Secondly, growth and ROC production by the six isolates was assessed by two are unavailable or are present at concentrations low enough to limit growth, many different compounds can be used as secondary nitrogen sources (i.e. nitrate, nitrite, most amino acids, peptides and proteins) (Garcia-Rico et al. 2009;Li et al. 2007). ...
... The P. roqueforti s.s. species has evolved to this simpler ROC gene cluster under natural conditions, not determined by recent industrial cheese-making practices(Garcia-Rico et al. 2008;Garcia-Rico et al. 2009; Kosalkova et al. 2015). ...
On Belgian farms, visible fungal growth is regularly encountered in ensiled feed commodities. The toxigenic fungal species Penicillium roqueforti sensu strictu (s.s.) and P. paneum, designated together as P. roqueforti sensu lato (s.l.) in this dissertation, are the most frequently isolated fungi in silages. Since the inhalation of fungal spores as well as the consumption of mycotoxin contaminated feed comprise serious health risks, it is of the outmost importance to prevent fungal contamination of silages. In this dissertation, general preventory measures are described. The final goal of this PhD research was to contribute to the prevention of specifically P. roqueforti s.l. development in silage. To achieve this goal, multiple in vitro lab experiments and in vivo trials with microsilos have been conducted, evaluating the effect of several abiotic and biotic factors on P. roqueforti s.l. growth and mycotoxin production. Roquefortine C, a mycotoxin that can be produced by both P. roqueforti s.s. and P. paneum, is considered to be an indicator of mycotoxin production by P. roqueforti s.l. in silages. Therefore, this particular mycotoxin has been determined to evaluate mycotoxin production. During the ensiling process, lactic acid bacteria convert sugars to mainly lactic acid, but also some acetic acid, methanol, ethanol, etc. These compounds can be used by P. roqueforti s.l. as a carbon source. Lactic acid as sole carbon source was not very conducive for fungal growth, while acetic acid (inhibiting aerobic deterioration and subsequent fungal development in silages) as sole carbon source facilitated good fungal growth. This illustrates that P. roqueforti s.l. is very well adapted to its silage-habitat, rendering prevention difficult. The bacterium Bacillus velezensis displayed antagonistic properties towards P. roqueforti s.l. in an in vitro experiment: both culture supernatant as cell suspension reduced spore germination and spore survival and inhibited fungal growth, without triggering an increased roquefortine C production. These observations seemed promising towards the capability of B. velezensis to prevent P. roqueforti s.l. development in silages, but the applied cell suspension could not live up to the great expectations regarding antagonism in an in vivo microsilo trial. Future research is required to investigate the potential of B. velezensis as a silage additive to counter P. roqueforti s.l. in silage. Oxygen appears to play a crucial role in the development of P. roqueforti s.l.: in anaerobic conditions, no fungal growth can occur. An in vivo microsilo trial with artificially infected whole-crop maize (@ 1500 conidia per gram fresh matter) desiled after 50, 100 or 150 days demonstrated that at desiling after 50 days some P. roqueforti s.l. propagules (66 per gram verse stof) had survived, while after an ensiled period of 100 days no active P. roqueforti s.l. propagules were detected. This experiment emphasizes the importance of a sufficiently long ensiled period, during which the integrity of the silo coverage needs to be maintained. In order to prevent the development of P. roqueforti s.l. in silages, the strict application of good agricultural practices regarding ensiling and desiling, limiting air ingress into silages, is the key factor to success.
