Expanding the Toolbox for Genetic Manipulation in Pseudogymnoascus: RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in P. verrucosus.

Fungi belonging to the genus Pseudogymnoascus have garnered increasing attention in recent years. One of the members of the genus, P. destructans, has been identified as the causal agent of a severe bat disease. Simultaneously, the knowledge of Pseudogymnoascus species has expanded, in parallel with the increased availability of genome sequences. Moreover, Pseudogymnoascus exhibits great potential as a producer of specialized metabolites, displaying a diverse array of biological activities. Despite these significant advancements, the genetic landscape of Pseudogymnoascus remains largely unexplored due to the scarcity of suitable molecular tools for genetic manipulation. In this study, we successfully implemented RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption in Pseudogymnoascus, using an Antarctic strain of Pseudogymnoascus verrucosus as a model. Both methods were applied to target azpA, a gene involved in red pigment biosynthesis. Silencing of the azpA gene to levels of 90% or higher eliminated red pigment production, resulting in transformants exhibiting a white phenotype. On the other hand, the CRISPR/Cas9 system led to a high percentage (73%) of transformants with a one-nucleotide insertion, thereby inactivating azpA and abolishing red pigment production, resulting in a white phenotype. The successful application of RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption represents a significant advancement in Pseudogymnoascus research, opening avenues for comprehensive functional genetic investigations within this underexplored fungal genus.

PrlaeA Affects the Production of Roquefortine C, Mycophenolic Acid, and Andrastin A in Penicillium roqueforti, but It Has Little Impact on Asexual Development.

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.

CRISPR/Cas9-Mediated Disruption of the pcz1 Gene and Its Impact on Growth, Development, and Penicillin Production in Penicillium rubens.

Penicillium rubens is a filamentous fungus of great biotechnological importance due to its role as an industrial producer of the antibiotic penicillin. However, despite its significance, our understanding of the regulatory mechanisms governing biological processes in this fungus is still limited. In fungi, zinc finger proteins containing a Zn(II)2Cys6 domain are particularly interesting regulators. Although the P. rubens genome harbors many genes encoding proteins with this domain, only two of them have been investigated thus far. In this study, we employed CRISPR-Cas9 technology to disrupt the pcz1 gene, which encodes a Zn(II)2Cys6 protein in P. rubens. The disruption of pcz1 resulted in a decrease in the production of penicillin in P. rubens. This decrease in penicillin production was accompanied by the downregulation of the expression of pcbAB, pcbC and penDE genes, which form the biosynthetic gene cluster responsible for penicillin production. Moreover, the disruption of pcz1 also impacts on asexual development, leading to decreased growth and conidiation, as well as enhanced conidial germination. Collectively, our results indicate that pcz1 acts as a positive regulator of penicillin production, growth, and conidiation, while functioning as a negative regulator of conidial germination in P. rubens. To the best of our knowledge, this is the first report involving a gene encoding a Zn(II)2Cys6 protein in the regulation of penicillin biosynthesis in P. rubens.

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (ß/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-ß-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (ß/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (ß/α)8-barrel domain.

Description of the First Four Species of the Genus Pseudogymnoascus From Antarctica.

The genus Pseudogymnoascus represents a diverse group of fungi widely distributed in different cold regions on Earth. Our current knowledge of the species of Pseudogymnoascus is still very limited. Currently, there are only 15 accepted species of Pseudogymnoascus that have been isolated from different environments in the Northern Hemisphere. In contrast, species of Pseudogymnoascus from the Southern Hemisphere have not yet been described. In this work, we characterized four fungal strains obtained from Antarctic marine sponges. Based on multilocus phylogenetic analyses and morphological characterizations we determined that these strains are new species, for which the names Pseudogymnoascus antarcticus sp. nov., Pseudogymnoascus australis sp. nov., Pseudogymnoascus griseus sp. nov., and Pseudogymnoascus lanuginosus sp. nov. are proposed. Phylogenetic analyses indicate that the new species form distinct lineages separated from other species of Pseudogymnoascus with strong support. The new species do not form sexual structures and differ from the currently known species mainly in the shape and size of their conidia, the presence of chains of arthroconidia, and the appearance of their colonies. This is the first report of new species of Pseudogymnoascus not only from Antarctica but also from the Southern Hemisphere.

Nonionizing Electromagnetic Field: A Promising Alternative for Growing Control Yeast.

In the food industry, some fungi are considered to be common spoilage microorganisms which reduce the shelf life of products. To avoid this outcome, different technologies are being developed to control their growth. Electromagnetic fields (EMF) have been used to combat bacterial growth, but there are few studies on yeasts and their possible action mechanisms. For this reason, we studied the effect of EMF between 1 to 5.9 GHz bands on the growth of Saccharomyces cerevisiae yeast and observed that all the frequencies of the band used cause the reduction of the viability of this yeast. In addition, we observed that the distance between the antenna and the sample is an important factor to consider to control the growing yeast. By using transmission electron microscopy, we found that the EMF caused a loss of continuity of the yeast cell membrane. Therefore, EMF may be used as a control method for yeast growth.

IMA genome - F14 : Draft genome sequences of Penicillium roqueforti, Fusarium sororula, Chrysoporthe puriensis, and Chalaropsis populi.

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.

Genetic Transformation of the Filamentous Fungus Pseudogymnoascus verrucosus of Antarctic Origin.

Cold-adapted fungi isolated from Antarctica, in particular those belonging to the genus Pseudogymnoascus, are producers of secondary metabolites with interesting bioactive properties as well as enzymes with potential biotechnological applications. However, at genetic level, the study of these fungi has been hindered by the lack of suitable genetic tools such as transformation systems. In fungi, the availability of transformation systems is a key to address the functional analysis of genes related with the production of a particular metabolite or enzyme. To the best of our knowledge, the transformation of Pseudogymnoascus strains of Antarctic origin has not been achieved yet. In this work, we describe for the first time the successful transformation of a Pseudogymnoascus verrucosus strain of Antarctic origin, using two methodologies: the polyethylene glycol (PEG)-mediated transformation, and the electroporation of germinated conidia. We achieved transformation efficiencies of 15.87 ± 5.16 transformants per µg of DNA and 2.67 ± 1.15 transformants per µg of DNA for PEG-mediated transformation and electroporation of germinated conidia, respectively. These results indicate that PEG-mediated transformation is a very efficient method for the transformation of this Antarctic fungus. The genetic transformation of Pseudogymnoascus verrucosus described in this work represents the first example of transformation of a filamentous fungus of Antarctic origin.

Heterologous expression, purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6-7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20-30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.

Cold-active pectinolytic activity produced by filamentous fungi associated with Antarctic marine sponges.

BACKGROUND: Pectinase enzymes catalyze the breakdown of pectin, a key component of the plant cell wall. At industrial level, pectinases are used in diverse applications, especially in food-processing industry. Currently, most of the industrial pectinases have optimal activity at mesophilic temperatures. On the contrary, very little is known about the pectinolytic activities from organisms from cold climates such as Antarctica. In this work, 27 filamentous fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new source of cold-active pectinases. RESULTS: In semi-quantitative plate assays, 8 out 27 of these isolates showed pectinolytic activities at 15 °C and one of them, Geomyces sp. strain F09-T3-2, showed the highest production of pectinases in liquid medium containing pectin as sole carbon source. More interesting, Geomyces sp. F09-T3-2 showed optimal pectinolytic activity at 30 °C, 10 °C under the temperature of currently available commercial mesophilic pectinases. CONCLUSION: Filamentous fungi associated with Antarctic marine sponges are a promising source of pectinolytic activity. In particular, pectinases from Geomyces sp. F09-T3-2 may be potentially suitable for biotechnological applications needing cold-active pectinases. To the best of our knowledge, this is the first report describing the production of pectinolytic activity from filamentous fungi from any environment in Antarctica.

The developmental regulator Pcz1 affects the production of secondary metabolites in the filamentous fungus Penicillium roqueforti.

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.

Cold-active pectinolytic activity produced by filamentous fungi associated with Antarctic marine sponges

BACKGROUND: Pectinase enzymes catalyze the breakdown of pectin, a key component of the plant cell wall. At industrial level, pectinases are used in diverse applications, especially in food-processing industry. Currently, most of the industrial pectinases have optimal activity at mesophilic temperatures. On the contrary, very little is known about the pectinolytic activities from organisms from cold climates such as Antarctica. In this work, 27 filamentous fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new source of cold-active pectinases. RESULTS: In semi-quantitative plate assays, 8 out 27 of these isolates showed pectinolytic activities at 15 °C and one of them, Geomyces sp. strain F09-T3-2, showed the highest production of pectinases in liquid medium containing pectin as sole carbon source. More interesting, Geomyces sp. F09-T3-2 showed optimal pectinolytic activity at 30 °C, 10 °C under the temperature of currently available commercial mesophilic pectinases. CONCLUSION: Filamentous fungi associated with Antarctic marine sponges are a promising source of pectinolytic activity. In particular, pectinases from Geomyces sp. F09-T3-2 may be potentially suitable for biotechnological applications needing cold-active pectinases. To the best of our knowledge, this is the first report describing the production of pectinolytic activity from filamentous fungi from any environment in Antarctica.

Role of sfk1 Gene in the Filamentous Fungus Penicillium roqueforti.

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.

Heterotrimeric G protein alpha subunit controls growth, stress response, extracellular protease activity, and cyclopiazonic acid production in Penicillium camemberti.

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 P. camemberti.

The Biosynthetic Gene Cluster for Andrastin A in Penicillium roqueforti.

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.

Identification and Functional Analysis of the Mycophenolic Acid Gene Cluster of Penicillium roqueforti.

The filamentous fungus Penicillium roqueforti is widely known as the ripening agent of blue-veined cheeses. Additionally, this fungus is able to produce several secondary metabolites, including the meroterpenoid compound mycophenolic acid (MPA). Cheeses ripened with P. roqueforti are usually contaminated with MPA. On the other hand, MPA is a commercially valuable immunosuppressant. However, to date the molecular basis of the production of MPA by P. roqueforti is still unknown. Using a bioinformatic approach, we have identified a genomic region of approximately 24.4 kbp containing a seven-gene cluster that may be involved in the MPA biosynthesis in P. roqueforti. Gene silencing of each of these seven genes (named mpaA, mpaB, mpaC, mpaDE, mpaF, mpaG and mpaH) resulted in dramatic reductions in MPA production, confirming that all of these genes are involved in the biosynthesis of the compound. Interestingly, the mpaF gene, originally described in P. brevicompactum as a MPA self-resistance gene, also exerts the same function in P. roqueforti, suggesting that this gene has a dual function in MPA metabolism. The knowledge of the biosynthetic pathway of MPA in P. roqueforti will be important for the future control of MPA contamination in cheeses and the improvement of MPA production for commercial purposes.

The pcz1 gene, which encodes a Zn(II)2Cys6 protein, is involved in the control of growth, conidiation, and conidial germination in the filamentous fungus Penicillium roqueforti.

Proteins containing Zn(II)(2)Cys(6) domains are exclusively found in fungi and yeasts. Genes encoding this class of proteins are broadly distributed in fungi, but few of them have been functionally characterized. In this work, we have characterized a gene from the filamentous fungus Penicillium roqueforti that encodes a Zn(II)(2)Cys(6) protein, whose function to date remains unknown. We have named this gene pcz1. We showed that the expression of pcz1 is negatively regulated in a P. roqueforti strain containing a dominant active Gαi protein, suggesting that pcz1 encodes a downstream effector that is negatively controlled by Gαi. More interestingly, the silencing of pcz1 in P. roqueforti using RNAi-silencing technology resulted in decreased apical growth, the promotion of conidial germination (even in the absence of a carbon source), and the strong repression of conidiation, concomitant with the downregulation of the genes of the central conidiation pathway brlA, abaA and wetA. A model for the participation of pcz1 in these physiological processes in P. roqueforti is proposed.