De Novo Peroxisome Biogenesis in Penicillium Chrysogenum Is Not Dependent on the Pex11 Family Members or Pex16

We have analyzed the role of the three members of the Pex11 protein family in peroxisome formation in the filamentous fungus Penicillium chrysogenum. Two of these, Pex11 and Pex11C, are components of the peroxisomal membrane, while Pex11B is present at the endoplasmic reticulum. We show that Pex11 is a major factor involved in peroxisome proliferation. We also demonstrate that P. chrysogenum cells deleted for known peroxisome fission factors (all Pex11 family proteins and Vps1) still contain peroxisomes. Interestingly, we find that, unlike in mammals, Pex16 is not essential for peroxisome biogenesis in P. chrysogenum, as partially functional peroxisomes are present in a pex16 deletion strain. We also show that Pex16 is not involved in de novo biogenesis of peroxisomes, as peroxisomes were still present in quadruple Δpex11 Δpex11B Δpex11C Δpex16 mutant cells. By contrast, pex3 deletion in P. chrysogenum led to cells devoid of peroxisomes, suggesting that Pex3 may function independently of Pex16. Finally, we demonstrate that the presence of intact peroxisomes is important for the efficiency of ß-lactam antibiotics production by P. chrysogenum. Remarkably, distinct from earlier results with low penicillin producing laboratory strains, upregulation of peroxisome numbers in a high producing P. chrysogenum strain had no significant effect on penicillin production

NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

Peroxisomes Are Required for Efficient Penicillin Biosynthesis in Penicillium chrysogenum▿ †

In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the side-chain precursor activation enzyme phenylacetyl CoA ligase (PCL), are crucial for efficient PEN synthesis. Moreover, increasing PEN titers are associated with increasing peroxisome numbers. However, not all conditions that result in enhanced peroxisome numbers simultaneously stimulate PEN production. We find that conditions that lead to peroxisome proliferation but simultaneously interfere with the normal physiology of the cell may be detrimental to antibiotic production. We furthermore show that peroxisomes develop in germinating conidiospores from reticule-like structures. During subsequent hyphal growth, peroxisome proliferation occurs at the tip of the growing hyphae, after which the organelles are distributed over newly formed subapical cells. We observed that the organelle proliferation machinery requires the dynamin-like protein Dnm1

Deletion of <i>pex3</i> in <i>P. chrysogenum</i> results in cells completely devoid of peroxisomes.

<p><i>P. chrysogenum</i> Δ<i>pex3</i> cells, producing GFP.SKL were grown for 40 h in PPM medium and analyzed by fluorescence (<b>A</b>) and electron (<b>B</b>) microscopy. Scale bars represent 5 µm in A and 1 µm in B; M-mitochondrion, N – nucleus.</p

Pex16 is not essential for peroxisome biogenesis.

<p>Fluorescence microscopy analysis of <i>P. chrysogenum</i> Δ<i>pex16</i> GFP.SKL (<b>A</b>) and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL (<b>B</b>) cells. Cells were grown for 40 h in PPM. For corresponding WT see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035490#pone-0035490-g003" target="_blank">Fig. 3A</a>. Scale bars represent 5 µm. <b>C</b>. Cells devoid of <i>pex16</i> are sporulation deficient. Colonies of WT and both Δ<i>pex16</i> GFP.SKL and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL strains were grown for 7 days on sporulation inducing R-agar plates. <b>D</b>. Δ<i>pex16</i> cells are characterized by decreased levels of Pex11. Western blots of WT and Δ<i>pex16</i> GFP.SKL cell extracts were prepared and decorated with α-Pex11 antibodies; translation elongation factor 1- (eEF1A) was used as a loading control. <b>E</b>. <i>P. chrysogenum</i> cells lacking <i>pex16</i> are able to grow on oleic acid although at decreased rates. WT, Δ<i>pex16</i> GFP.SKL and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL strains were grown for 10 days on mineral medium containing 0.5% glucose or 0.1% oleic acid as a sole carbon source. Electron micrographs of Δ<i>pex16</i> GFP.SKL (<b>F</b>) and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL (<b>G</b>) cells grown for 40 h in PPM; P – peroxisome; M – mitochondrion; V – vacuole; arrows indicate protein dense inclusions. Scale bars represent 1 µm. Electron micrographs representing α-IAT immunolabelling of sections of Δ<i>pex16</i> GFP.SKL (<b>H</b>) and Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> Δ<i>pex16</i> GFP.SKL (<b>I</b>) cells. Scale bars represent 1 µm.</p

The effect of overproduction of Pex11 family members on peroxisome proliferation.

<p>Representative CLSM images of <i>P. chrysogenum</i> GFP.SKL cells: WT (<b>A</b>); overproducing Pex11 (<b>B</b>), Pex11B (<b>C</b>) or Pex11C (<b>D</b>). Cells were grown for 40 h in PPM. Scale bars represent 5 µm. <b>E</b>. Western blot showing overproduction of Pex11 (top), Pex11B, (center) or Pex11C (bottom). Crude extracts of DS17690 and strains overproducing Pex11 proteins were used for SDS-PAGE and western blotting and probed with specific antisera. Equal amounts of protein were loaded per lane. <b>F</b>. Electron micrograph of <i>P. chrysogenum</i> cells overproducing Pex11B. P-peroxisome; M-mitochondrion. Scale bar represents 1 µm.</p

<i>P. chrysogenum</i> cells devoid of all Pex11 family members still contain peroxisomes.

<p>Double and triple deletion mutants of <i>pex11</i> family genes were prepared and analyzed by CLSM after growth for 40 h in PPM: WT (<b>A</b>), <i>Δpex11</i> (<b>B</b>), <i>Δpex11</i> Δ<i>pex11B</i> GFP.SKL (<b>C</b>), Δ<i>pex11</i> Δ<i>pex11C</i> GFP.SKL (<b>D</b>), Δ<i>pex11B</i> Δ<i>pex11C</i> GFP.SKL (<b>E</b>), Δ<i>pex11</i> Δ<i>pex11B</i> Δ<i>pex11C</i> GFP.SKL (<b>F</b>). Scale bars represent 5 µm.</p

Impact of deletion of <i>pex11</i> family genes, <i>pex16</i> or <i>pex3</i> on penicillin production.

<p><b>A.</b> Analysis of the production of antibacterial compounds by selected strains using plate bioassays with <i>M. luteus</i> as an indicator strain. Clarified culture supernatants were diluted 3200 times before analysis. During each experiment a corresponding WT supernatant at the same dilution was tested on the same plate as the supernatants of the analyzed mutants. <b>B and C.</b> Western blot analysis of the levels of penicillin biosynthetic enzymes IPNS and IAT in strains with manipulated levels of Pex11 family proteins (<b>B</b>), Pex3 or Pex16 (<b>C</b>). eEF1A was used as a loading control.</p

Subcellular localization of Pex11 family members.

<p><i>P. chrysogenum</i> cells producing C-terminal mGFP fusions of Pex11 (<b>A</b>), Pex11C (<b>B</b>) or Pex11B (<b>C</b>) with DsRed.SKL as peroxisome marker were grown for 40 h in PPM and analyzed by CLSM. <b>D</b>. CLSM analysis of <i>P. chrysogenum</i> hyphae producing Pex11B.mGFP and Sec63.mCherry as marker of the ER, grown for 40 h in PPM. Scale bars represent 5 µm. Arrowheads (in <b>C</b>) indicate the sites of overlap between Pex11B.mGFP and Sec63.mCherry fluorescence.</p

<i>P. chrysogenum</i> Pex16 is a peroxisomal membrane protein.

<p><i>P. chrysogenum</i> cells producing Pex16.mGFP and either DsRed.SKL (<b>A</b>) or Sec63.mCherry (<b>B</b>) were grown for 40 h in PPM and analyzed by fluorescence microscopy. Scale bars represent 5 µm.</p