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)(2)Cys(6) 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.This work was supported by project Fondecyt 1120833 and "Proyecto DICYT, Codigo 021743CR, Vicerrectoria de Investigation, Desarrollo e Innovation, Universidad de Santiago de Chile", and MIISSB Iniciativa Cientifica Milenio-MINECON. JFR-A and CG-D have received doctoral fellowships CONICYT-PFCHA/Doctorado National/2013-21130251 and CONICYT-PFCHA/Doctorado National/2014-63140056, respectively

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

The biosynthetic gene cluster for andrastin A in Penicillium roqueforti

© 2017 Rojas-Aedo, Gil-Durán, Del-Cid, Valdés, álamos, Vaca, García-Rico, Levicán, Tello and Chávez. 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 s

Role of sfk1 gene in the filamentous fungus Penicillium roqueforti

© 2017 Torrent, Gil-Durán, Rojas-Aedo, Medina, Vaca, Castro, García-Rico, Cotoras, Mendoza, Levicán and Chávez. 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 als

qRT-PCR analysis of the expression of <i>mpa</i> genes in RNAi-silenced transformants of <i>P</i>. <i>roqueforti</i>.

<p>The RNAi-silenced transformants selected were T4 and T7 for <i>mpaA</i>, T1 and T7 for <i>mpaB</i>, T5 and T6 for <i>mpaC</i>, T1 and T2 for <i>mpaDE</i>, T4 and T6 for <i>mpaF</i>, T2 and T3 for <i>mpaG</i>, and T5 and T6 for <i>mpaH</i>. Total RNA extractions and qRT-PCR experiments were conducted as described in Materials and Methods. Wild-type <i>P</i>. <i>roqueforti</i> CECT 2905 (WT) and <i>P</i>. <i>roqueforti</i> CECT 2905 containing empty pJL43-RNAi vector (E) were used as controls. Error bars represent the standard deviation of three replicates in three different experiments. The differences were considered statistically significant at <i>P</i> < 0.05 (*) using Student’s <i>t</i>-test.</p

HPLC analysis of the known intermediates of the MPA biosynthesis pathway in RNAi-silenced transformants of <i>P</i>. <i>roqueforti</i>.

<p>(A) Accumulation of DHMP in the <i>mpaA</i> silenced transformant T7. (B) Accumulation of 5-MOA in the <i>mpaDE</i> silenced transformant T2. (C) Accumulation of DMMPA in the <i>mpaG</i> silenced transformant T3. The HPLC trace chromatograms (300 nm) of the transformants are shown as red line, whereas the trace chromatogram (300 nm) of the wild-type strain (control) is shown as black line. In each panel, the peak representing the accumulated compound is indicated by an arrow. The identity of each peak was assigned based on the expected retention time and its UV absorption spectrum (shown in the inset of each panel). The wavelengths of each UV absorption maximum (nm) are 216, 257 and 296 for DHMP; 217, 260 and 296 for 5-MOA; 216, 258 and 304 for DMMPA. These values are in agreement with those described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147047#pone.0147047.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147047#pone.0147047.ref029" target="_blank">29</a>]. AU: Absorbance units.</p

Schematic organization of the MPA biosynthetic gene cluster in <i>P</i>. <i>roqueforti</i>.

<p>The arrows indicate the genes and the direction of their transcription. The correspondence of each <i>mpa</i> gene with their respective ORFs previously annotated in the draft genome of <i>P</i>. <i>roqueforti</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147047#pone.0147047.ref018" target="_blank">18</a>] is indicated in the boxes. According to our analysis, <i>mpaB</i> should be longer than ORF Proq05g069810, so this ORF would partially contain <i>mpaB</i>. In contrast, Proq05g069780a is longer than the <i>mpaDE</i> gene predicted by us.</p

Production of MPA by <i>P</i>. <i>roqueforti</i> (WT) and RNAi-silenced transformants.

<p>Transformants are the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147047#pone.0147047.g002" target="_blank">Fig 2</a>. Metabolites were extracted from mycelium and quantified as described in Materials and Methods. In each case, the quantity of MPA was normalized by the dry weight of the fungal mycelia extracted. Error bars represent the standard deviation of three replicates in three independent experiments. Statistical analysis using Student’s <i>t</i>-test (P < 0.05) indicates significant differences between the production of MPA by the wild-type strain (*) and the transformants. Please note that MPA production of <i>P</i>. <i>roqueforti</i> containing empty pJL43-RNAi vector was statistically indistinguishable from the wild-type strain. When MPA was extracted from agar, the results were similar (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147047#pone.0147047.s008" target="_blank">S8 Fig</a>).</p

Sensitivity towards MPA of transformant T6, with attenuated expression of <i>mpaF</i>, and wild-type <i>P</i>. <i>roqueforti</i>.

<p>Ten-fold serial dilutions (10⁶ to 10) of spores from T6 and the wild-type strain (WT) were spotted on CYA plates with (+MPA) or without (-MPA) 100 μg MPA/ml. Please note the reduction in the germination of spores of transformant T6 in presence of MPA, especially evident at dilutions 10⁴ and 10³, compared with the wild-type strain.</p