An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets.

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied-one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.

Penicillium chrysogenum as a model system for studying cellular effects of methylglyoxal.

BACKGROUND: α-oxoaldehydes are formed as toxic by-products during metabolic activity. The biologically most important compound of this class, methylglyoxal, results from spontaneous phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate which are intermediate glycolysis products. Methylglyoxal-mediated modification of lipids, nucleic acids and proteins is known to lead to the formation of advanced glycation end products. These modifications contribute to the aetiology of severe diseases like diabetes and neurodegenerative disorders. By using simple model organisms it is possible to conveniently study the effects of methylglyoxal on cellular processes. Here, results are presented on the effects of methylglyoxal on mycelium growth, stationary phase entry (monitored by autophagy induction), mitochondrial morphology and protein composition in the filamentous fungus Penicillium chrysogenum. RESULTS: Methylglyoxal leads to growth rate reduction of this fungus so that the entry into the stationary phase is delayed. Mitochondrial morphology is not changed by methylglyoxal. However, rapamycin-mediated fragmentation of mitochondria is prevented by methylglyoxal. Furthermore, three proteins are identified that are present in lower abundance when methylglyoxal is added to the growth medium (aldo-keto reductase [Pc22g04850], 5-methyl-tetrahydropteroyl-triglutamate-homocysteine S-methyltransferase [Pc22g18630] and NAD-dependent formate dehydrogenase [Pc12g04310]). CONCLUSIONS: The presented results contribute to the understanding of cellular pathways and mechanisms that are affected by the ubiquitous α-oxoaldehyde methylglyoxal.

Genome-wide identification of target genes of a mating-type α-domain transcription factor reveals functions beyond sexual development.

Penicillium chrysogenum is the main industrial producer of the ß-lactam antibiotic penicillin, the most commonly used drug in the treatment of bacterial infections. Recently, a functional MAT1-1 locus encoding the α-box transcription factor MAT1-1-1 was discovered to control sexual development in P. chrysogenum. As only little was known from any organism about the regulatory functions mediated by MAT1-1-1, we applied chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) to gain new insights into the factors that influence MAT1-1-1 functions on a molecular level and its role in genome-wide transcriptional regulatory networks. Most importantly, our data provide evidence for mating-type transcription factor functions that reach far beyond their previously understood role in sexual development. These new roles include regulation of hyphal morphology, asexual development, as well as amino acid, iron, and secondary metabolism. Furthermore, in vitro DNA-protein binding studies and downstream analysis in yeast and P. chrysogenum enabled the identification of a MAT1-1-1 DNA-binding motif, which is highly conserved among euascomycetes. Our studies pave the way to a more general understanding of these master switches for development and metabolism in all fungi, and open up new options for optimization of fungal high production strains.

Submicronic fungal bioaerosols: high-resolution microscopic characterization and quantification.

Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min(-1) airflow. The overall median numbers of aerosolized submicronic particles were 2 × 10(5) cm(-2), 2.6 × 10(3) cm(-2), and 0.9 × 10(3) cm(-2) for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.

Effect of low shear modeled microgravity on phenotypic and central chitin metabolism in the filamentous fungi Aspergillus niger and Penicillium chrysogenum.

Phenotypic and genotypic changes in Aspergillus niger and Penicillium chrysogenum, spore forming filamentous fungi, with respect to central chitin metabolism were studied under low shear modeled microgravity, normal gravity and static conditions. Low shear modeled microgravity (LSMMG) response showed a similar spore germination rate with normal gravity and static conditions. Interestingly, high ratio of multiple germ tube formation of A. niger in LSMMG condition was observed. Confocal laser scanning microscopy images of calcofluor flurophore stained A. niger and P. chrysogenum showed no significant variations between different conditions tested. Transmission electron microscopy images revealed number of mitochondria increased in P. chrysogenum in low shear modeled microgravity condition but no stress related-woronin bodies in fungal hyphae were observed. To gain additional insight into the cell wall integrity under different conditions, transcription level of a key gene involved in cell wall integrity gfaA, encoding the glutamine: fructose-6-phosphate amidotransferase enzyme, was evaluated using qRT-PCR. The transcription level showed no variation among different conditions. Overall, the results collectively indicate that the LSMMG has shown no significant stress on spore germination, mycelial growth, cell wall integrity of potentially pathogenic fungi, A. niger and P. chrysogenum.

Compartmentalization in penicillin G biosynthesis by Penicillium chrysogenum PQ-96.

The arrangement of organelles in the sub-apical productive non-growing vacuolated hyphal cells of the high- and the low-penicillin-pro- ducing strains Penicillium chrysogenum was compared using transmission electron microscopy. In the productive cells of the high-yielding strain the endoplasmic reticulum and the polyribosomes with associated peroxisomes are frequently arranged at the periphery of the cytoplasm and around the vacuoles. At the high activity of penicillin G biosynthesis the immuno-label of the cytosolic isopenicillin N synthase is concentrated at the polyribosomes arranged in the peripheral cytoplasm and along the tonoplast as well as around the peroxisomes. On the basis of the obtained results the compartmentalization of the pathway of penicillin G biosymthesis is discussed. The obtained results support the phenylacetic acid detoxification hypothesis of penicillin G biosynthesis.

Effect of fungi and light on seed germination of three Opuntia species from semiarid lands of central Mexico.

Fungal attack under light reduces mechanical resistance of the testa of Opuntia seeds, making it easier for the embryo to emerge. However, the effect of fungi on Opuntia seed germination in darkness is unknown. We evaluated the combined effects of light and inoculation with Phoma medicaginis, Trichoderma harzianum, Trichoderma koningii, and Penicillium chrysogenum on germination of O. streptacantha, O. leucotricha, and O. robusta seeds, from central Mexico. We also evaluated the combined effects of seed age (2-, 3-, and 12-year-old seeds) and presence of fungi on the testa on O. streptacantha germination. All fungal species eroded the funicular envelope and promoted seed germination for O. leucotricha and O. streptacantha, but did more so in light than in darkness. For the latter species, younger seeds inoculated with fungi had lower germination than older ones. For O. robusta, we found that seeds inoculated with P. medicaginis and T. harzianum had similar germination in light and in darkness. Our results strongly indicate that deterioration of the testa by fungi is higher in light than in darkness.

Bioleaching mechanism of heavy metals in the mixture of contaminated soil and slag by using indigenous Penicillium chrysogenum strain F1.

The ability and bioleaching mechanism of heavy metals by Penicillium chrysogenum in soils contaminated with smelting slag were examined in this study. Batch experiments were performed to investigate the growth kinetics of P. chrysogenum, organic acids production and to compare the removal efficiencies of heavy metals between bioleaching with P. chrysogenum and chemical organic acids. The results showed that the bioleaching had higher removals than chemical leaching, and the removal percentages of Cd, Cu, Pb, Zn, Mn and Cr reached up to 74%, 59%, 24%, 55%, 57% and 25%, respectively. Removal efficiencies of heavy metals (15.41 mg/50 mL) by bioleaching were higher than chemical leaching with 0.5% of citric acid (15.15 mg/50 mL), oxalic acid (8.46 mg/50 mL), malic acid (11.35 mg/50 mL) and succinic acid (10.85 mg/50 mL). The results of transmission electron microscope (TEM) showed that no damage was obviously observed on the surface of the living cell except for thinner cell wall, discontinuous plasma membrane, compartmentalized lumen and concentrated cytoplasm during bioleaching process. The activity of extracellular glucose oxidase (GOD) produced by P. chrysogenum is influenced severely by the multi-heavy metal ions. The result implied that P. chrysogenum can be used to remove heavy metals from polluted soil and smeltery slag.

Bioleaching of heavy metals from a contaminated soil using indigenous Penicillium chrysogenum strain F1.

Bioleaching of heavy metals from contaminated soil using Penicillium chrysogenum strain F1 was investigated. Batch experiments were performed to compare leaching efficiencies of heavy metals between one-step and two-step processes and to determine the transformation of heavy metal fractions before and after bioleaching. The results showed that two-step process had higher leaching efficiencies of heavy metals than one-step process. When the mass ratio of soil to culture medium containing P. chrysogenum strain F1 was 5% (w/v), 50%, 35%, 9% and 40% of Cd, Cu, Pb and Zn were removed in one-step process, respectively. The two-step process had higher removals of 63% Cd, 56% Cu, 14% Pb and 54% Zn as compared with one-step process. The results of the sequential extraction showed that the metals remaining in the soil were mainly bonded in stable fractions after bioleaching. The results of TEM and SEM showed that during bioleaching process, although the mycelium of P. chrysogenum was broken into fragments, no damage was obviously observed on the surface of the living cell except for thinner cell wall, smaller vacuoles and concentrated cytoplasm. The result implied that P. chrysogenum strain F1 can be used to remove heavy metals from polluted soil.

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.

Two components of a velvet-like complex control hyphal morphogenesis, conidiophore development, and penicillin biosynthesis in Penicillium chrysogenum.

Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here, we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, which we have named P. chrysogenum velA (PcvelA) and PclaeA. Data from array analysis using a DeltaPcvelA deletion strain indicate a significant role of PcVelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and high-performance liquid chromatography quantifications of penicillin titers clearly show that both PcVelA and PcLaeA play a major role in penicillin biosynthesis in a producer strain that underwent several rounds of UV mutagenesis during a strain improvement program. Both regulators are further involved in different developmental processes. While PcvelA deletion leads to light-independent conidial formation, dichotomous branching of hyphae, and pellet formation in shaking cultures, a DeltaPclaeA strain shows a severe impairment in conidiophore formation under both light and dark conditions. Bimolecular fluorescence complementation assays provide evidence for a velvet-like complex in P. chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators in genera other than Aspergillus.

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells.

In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.

Fungal biomass with grafted poly(acrylic acid) for enhancement of Cu(II) and Cd(II) biosorption.

The biomass of Penicillium chrysogenum was modified by graft polymerization of acrylic acid (AAc) on the surface of ozone-pretreated biomass. The sorption capacity for copper and cadmium increased significantly as a large number of carboxyl groups were present on the biomass surface, especially when the carboxylic acid group was converted to carboxylate ions using NaOH. When modeled using the Langmuir isotherm, the sorption capacities were 1.70 and 1.87 mmol g(-1) for copper and cadmium, respectively. The loaded biosorbent was regenerated using HCl solution and used repeatedly over five cycles with little loss of uptake capacity beyond the second cycle. The sorption of the two metals was time-dependent, and the kinetics fitted the pseudo-second-order equation well. The Freundlich, Langmuir, Temkin, and Dubinin-Redushkevich isotherms were used to model the metal sorption isotherms, and the thermodynamic parameters calculated show that the sorption was spontaneous and endothermic under the condition applied and that the biomass has similar sorption affinities for the two metals. Fourier transform infrared and X-ray photoelectron spectroscopy reveal that carboxyl, amide, and hydroxyl groups on the biomass surface were involved in the sorption of copper and cadmium and ion exchange and complexation dominated the sorption process.

Overproduction of a single protein, Pc-Pex11p, results in 2-fold enhanced penicillin production by Penicillium chrysogenum.

Current industrial production of beta-lactam antibiotics, using the filamentous fungus Penicillium chrysogenum, is the result of many years of strain improvement by classical mutagenesis. More efficient production strains showed significant increases in the number and volume fraction of microbodies in their cells, organelles that harbor key enzymes involved in the biosynthesis of beta-lactam antibiotics. We have isolated the P. chrysogenum cDNA encoding Pc-Pex11p, a peroxin that is involved in microbody abundance. We demonstrate that overproduction of Pc-Pex11p in P. chrysogenum results in massive proliferation of tubular-shaped microbodies and a 2- to 2.5-fold increase in the level of penicillin in the culture medium. Notably, Pc-Pex11p-overproduction did not affect the levels of the enzymes of the penicillin biosynthetic pathway. Our results suggest that the stimulating effect of enhanced organelle numbers may reflect an increase in the fluxes of penicillin and/or its precursors across the now much enlarged microbody membrane.

New insights into the target site and mode of action of the antifungal protein of Aspergillus giganteus.

The antifungal protein (AFP) secreted by Aspergillus giganteus exerts growth inhibitory effects on various filamentous fungi. In order to obtain more information on the mode of action of AFP, we used transmission electron microscopy in this study to compare the cellular ultrastructure of the AFP-sensitive Aspergillus niger and of the AFP-resistant Penicillium chrysogenum upon AFP treatment. Furthermore, AFP was localized by immunogold staining in both fungi. Severe membrane alterations in A. niger were observed, whereas the membrane of P. chrysogenum was not affected after treatment with AFP. The protein localized predominantly to a cell wall attached outer layer which is probably composed of glycoproteins, as well as to the cell wall of A. niger. It was found to accumulate within defined areas of the cell wall, pointing towards a specific interaction of AFP with cell wall components. In contrast, very little protein was bound to the outer layer and cell wall of P. chrysogenum. For future applications of AFP as an antimycotic drug, the mode of action of the protein was further characterized. The protein was found to act in a dose-dependent manner: it was fungistatic when applied at concentrations below the minimal inhibitory concentration, but fungicidal at higher concentrations. Using an in vivo model system, we were able to finally show that AFP indeed prevented the infection of tomato roots (Lycopersicon esculentum) by the plant-pathogenic fungus Fusarium oxysporum f. sp. lycopersici.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme.

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteine, and L-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.

Assessment of the microbody luminal pH in the filamentous fungus Penicillium chrysogenum.

The enzymes of the penicillin biosynthetic pathway in Penicillium chrysogenum are located in different subcellular compartments. Consequently, penicillin pathway precursors and the biologically active penicillins have to cross one or more membranes. The final enzymatic step that is mediated by acyltransferase takes place in a microbody. The pH of the microbody lumen in penicillin producing cells has been determined with fluorescent probes and mutants of the green fluorescent protein and found to be slightly alkaline.

Isolation of Penicillium chrysogenum PEX1 and PEX6 encoding AAA proteins involved in peroxisome biogenesis.

In Penicillium chrysogenum, key enzymes involved in the production of penicillin reside in peroxisomes. As a first step to understand the role of these organelles in penicillin biosynthesis, we set out to isolate the genes involved in peroxisome biogenesis. Here we report the cloning and characterization of P. chrysogenum PEX1 and PEX6, which encode proteins of the AAA family of ATPases. The second AAA module, which is essential for the function of Pex1p and Pex6p in peroxisome biogenesis, is highly conserved in both PcPexlp and PcPex6p. PcPEX1 and PcPEX6 contain three and two introns, respectively.

Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.

A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10,000 x g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14,000 x g pellet. After 2 h, 45,000 x g isopycnic centrifugation of the 14,000 x g pellet on a continuous 20-60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.

Structural and functional properties of plasma membranes from the filamentous fungus Penicillium chrysogenum.

Functional plasma membranes from the filamentous fungus Penicillium chrysogenum have been isolated with the objective of studying transport processes. The isolation procedure consists of three steps, namely homogenization of cells with a Braun MSK homogenizer, followed by Percoll gradient centrifugation and floatation of membranes in a three-step Nycodenz gradient. This method can be applied to strains which differ significantly in morphology and penicillin-production capacity. Plasma membranes were fused with liposomes containing the beef heart mitochondrial cytochrome-c oxidase. In the presence of reduced cytochrome c, the hybrid membranes maintained a high proton motive force that functions as a driving force for the uptake of the amino acids arginine and valine via distinct transport systems.