Cold-active xylanase produced by fungi associated with Antarctic marine sponges

Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 C in semiquantitative plate assays. One of these isolates, Cladosporium sp.; showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5-7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s

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 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

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

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

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

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

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