Chrysogenum Strain Research Articles

Synthesis and Anti-microbial Activities of Some Thiourea Based Metal Complexes

Normal 0 The transition metal (II) complexes were formed by the reaction of Co(II), Ni(II), Zn(II), Fe (II) and Cu (II) nitrates with the schiff base ligands. The complexes were characterized using infrared (FT-IR), electronic spectral (UV), elemental analysis, melting points and XRD analysis etc. The transition metal (II) complexes were screened for antibacterial and antifungal activities. Antibacterial activity against four bacteria such as Escherichia coli, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis bacterial strains by the agar-cup method. Antifungal activity was studied against four bacteria Aspergillus Niger, Penicillium Chrysogenum, fusarium moneliforme and aspergillus flavus bacterial strains by the poison plate method. The complexes were found to exhibit higher to moderate activity against some bacterial species.

Structural variation among wild and industrial strains of Penicillium chrysogenum.

Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54–1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54–1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54–1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts.

Synthesis and Characterization of Aromatic-Heterocyclic Derivatives of Sulphonamides as Potential Antimicrobial Agents

The incidence of microbial infection is increasing world-wide despite active research on the discovery and development of novel anti-microbial agents. Then emergence of multi-resistant strains of bacteria has increased the problem of drug resistance dramatically. The development and use of new anti-microbial agents is one of the ways to combat the emergence of anti-microbial resistance. The literature survey reveals that the sulfonamide derivatives as privileged pharmacophore for wide range of biological activities including anti-microbial activity. So a series of (3a-3e and 6a-6c) sulfonamide derivatives were synthesized by attaching alkyl/ aryl/heterocyclic sulphonyl chloride and alkyl/aryl carbonyl chloride moieties to aromatic sulfonamide incorporating free amino group by Schotten Baumann (SB) reaction. All the synthesized compounds were characterized by FTIR, Mass, and other analytical techniques. The synthesized compounds have been screened against different bacterial (B. subtilis, S. aureus and E. coli) and fungal (A. niger and P. chrysogenum) strains using the cup-plate method. The synthesized sulfonamides derivatives showed significant antibacterial activity against Gram-positive and Gram-negative organisms, while they were found to be inactive against fungal strains. From the results, it is evident that the presence of a hetero/aromatic ring showed good antibacterial activity. Some of these compounds can be considered as potential candidates for treatment of infections caused by Gram-positive as well as Gram-negative bacteria.

Genomic characteristics and comparative genomics analysis of Penicillium chrysogenum KF-25

BackgroundPenicillium chrysogenum has been used in producing penicillin and derived β-lactam antibiotics for many years. Although the genome of the mutant strain P. chrysogenum Wisconsin 54-1255 has already been sequenced, the versatility and genetic diversity of this species still needs to be intensively studied. In this study, the genome of the wild-type P. chrysogenum strain KF-25, which has high activity against Ustilaginoidea virens, was sequenced and characterized.ResultsThe genome of KF-25 was about 29.9 Mb in size and contained 9,804 putative open reading frames (orfs). Thirteen genes were predicted to encode two-component system proteins, of which six were putatively involved in osmolarity adaption. There were 33 putative secondary metabolism pathways and numerous genes that were essential in metabolite biosynthesis. Several P. chrysogenum virus untranslated region sequences were found in the KF-25 genome, suggesting that there might be a relationship between the virus and P. chrysogenum in evolution. Comparative genome analysis showed that the genomes of KF-25 and Wisconsin 54-1255 were highly similar, except that KF-25 was 2.3 Mb smaller. Three hundred and fifty-five KF-25 specific genes were found and the biological functions of the proteins encoded by these genes were mainly unknown (232, representing 65%), except for some orfs encoding proteins with predicted functions in transport, metabolism, and signal transduction. Numerous KF-25-specific genes were found to be associated with the pathogenicity and virulence of the strains, which were identical to those of wild-type P. chrysogenum NRRL 1951.ConclusionGenome sequencing and comparative analysis are helpful in further understanding the biology, evolution, and environment adaption of P. chrysogenum, and provide a new tool for identifying further functional metabolites.

Development of a multiplex qPCR method for simultaneous quantification in dry-cured ham of an antifungal-peptide Penicillium chrysogenum strain used as protective culture and aflatoxin-producing moulds

A multiplex qPCR method to quantify the implantation of the antifungal-peptide-producing Penicillium chrysogenum RP42C and aflatoxin-producing moulds including a non-competitive IAC in dry-cured ham was developed. For this, the primer pairs F/R-Pc, F/R-omt and Tub-F1/R1 and the TaqMan probes, P-Pc, OMTprobe and Tubprobe targeting the pgafp, omt-1 and β-tubulin genes, respectively, were used. The ability of the optimized qPCR method to quantify simultaneously both non-toxigenic and toxigenic moulds in inoculated dry-cured ham was demonstrated since efficiency values ranged from 102.1 to 111.1%, and the limit of detection was between 2 and 3 log cfu/cm2 for both antifungal-peptide- and aflatoxin-producing moulds. In addition, suitability of multiplex qPCR to test implantation of protective P. chrysogenum as well as growth of aflatoxigenic strain in a controlled model system was carried out successfully. The multiplex qPCR allowed demonstrating that the protective strain of P. chrysogenum RP42C limits growth of the aflatoxin-producing Aspergillus flavus strain on dry-cured ham. These findings were confirmed by the results of aflatoxin analysis, since aflatoxin B1 was not detected when the protective P. chrysogenum RP42C was inoculated. Furthermore, the multiplex qPCR including an IAC quantified efficiently the implantation of protective culture P. chrysogenum RP42C in dry-cured ham after 6 months of incubation. Thus, the multiplex qPCR should be considered a sensitive and rapid tool to monitor the implantation of fungal protective cultures and to determine growth of aflatoxin-producing moulds in dry-cured ham as well.

Fungal leaching of metals from electronic scrap

The present study was carried out to develop an economically feasible and environmentally friendly technique to extract metals such as Al, Fe, Cu, Zn and Ni from electronic scrap by employing Penicillium chrysogenum strain KBS3. Studies were carried out in various leaching modes, including one-step leaching with combined growth and leaching phases (Mode 1), two-step leaching with alternative growth and leaching phases (Mode 2), spent medium leaching with separated growth and leaching phases (Mode 3), leaching with fresh growth medium without fungal culture (Mode 4), leaching with deionized water (Mode 5) and leaching with commercial organic acids (Mode 6). The main lixiviants produced in leaching Mode 1 were citric, tartaric, oxalic and gluconic acids in concentrations of 12, 2.5, 1.8 and 152 mM, whereas in leaching Mode 2, the concentrations were 15, 0.5, 1.0 and 1,162 mM, respectively. In leaching Mode 3, 63 mM citric acid, 23 mM tartaric acid and 29 mM oxalic acid was observed. At 5% pidp density, both leaching Modes 1 and 2 showed similar metal extraction yields from the electronic scrap at 35° C. Approximately 96% Al, 98% Zn, 48% Cu, 25% Fe, 66% Pb and 73% Ni were extracted in leaching Mode 1 in 14 days. In leaching Mode 3, 85% Al, 81 % Zn, 97% Cu, 22% Fe, 50% Pb and 63% Ni were extracted in 14 days. The extraction yield of Cu was much lower in leaching Modes 1 and 2 (48–50%) than in leaching Mode 3 (97%), as well as in leaching Mode 6 (98%) with organic acids, including citric, tartaric, oxalic and gluconic acids.

Sweet Wine Production by Two Osmotolerant Saccharomyces cerevisiae Strains

The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts.

IMPROVEMENT OF P. CHRYSOGENUM BY UV MUTATION FOR OVERPRODUCTION OF CELLULASE AND LIGNINASE

The improvement of microbial strains for the overproduction of lignocellulolytic enzymes production by mutagenesis has attracted attention in the commercial fermentation industry. The present study was designed to modify a Penicillium chrysogenum strain, overproduce cellulase and ligninase, and see the relationship between exposure time and distance of delivering UV rays to fungal strains for the improvement of enzyme production.UV radiation source was placed above the surface of P. chrysogenum suspension spores. Radiation was delivered at 30W at various distances between 10cm ~ 50cm for 10 ~ 60mins intervals of 10min. Mutants obtained from the irradiation were selected and cultured in mineral salt media for enzyme assays. Reducing sugars released were determined as well as enzyme activity of the cellulase (FPase and CMCase) and ligninase (Laccase, MnP, LiP). Growths on plates were also monitored and studied.The FPase and CMCase activity of the mutant strains were two folds higher than that of the parent strains. Similar trend was observed with the LiP, MnP and Laccase where there was 5 fold, 15 fold, and 9.6 fold respectively.There was no significant difference in FPase activities (P>;0.05) for the different exposure time and distance from source of radiation, however, there was in the case of CMCase. There was no significant difference (P>;0.05) for Laccase and Manganese Peroxidase, however there was significant difference for Lignin peroxidase. The distance at which Ultraviolet Irradiation is delivered and the exposure time could significantly affect the mutation of fungi for production of lignocellulolytic enzymes.

Fungal Inoculation and Elevated CO2 Mediate Growth of Lolium Mutiforum and Phytolacca Americana, Metal Uptake, and Metal Bioavailability in Metal-Contaminated Soil: Evidence from DGT Measurement

Fungal inoculation and elevated CO2 may mediate plant growth and uptake of heavy metals, but little evidence from Diffusive Gradients in Thin-films (DGT) measurement has been obtained to characterize the process. Lolium mutiforum and Phytolacca americana were grown at ambient and elevated CO2 on naturally Cd and Pb contaminated soils inoculated with and without Trichoderma asperellum strain C3 or Penicillium chrysogenum strain D4, to investigate plant growth, metal uptake, and metal bioavailability responses. Fungal inoculation increased plant biomass and shoot/root Cd and Pb concentrations. Elevated CO2 significantly increased plants biomass, but decreased Cd and Pb concentrations in shoot/root to various extents, leading to a metal dilution phenomenon. Total Cd and Pb uptake by plants, and DGT-measured Cd and Pb concentrations in rhizosphere soils, were higher in all fungal inoculation and elevated CO2 treatments than control treatments, with the combined treatments having more influence than either treatment alone. Metal dilution phenomenon occurred because the increase in DGT-measured bioavailable metal pools in plant rhizosphere due to elevated CO2 was unable to match the increase in requirement for plant uptake of metals due to plant biomass increase.

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 (15.41mg/50mL) by bioleaching were higher than chemical leaching with 0.5% of citric acid (15.15mg/50mL), oxalic acid (8.46mg/50mL), malic acid (11.35mg/50mL) and succinic acid (10.85mg/50mL). 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.

Fungal Bioleaching of Metals From Mine Tailing

The present study was undertaken to develop an economically feasible and environmental-friendly technique to recover metals (Ni, Cu, Co, Zn, Mg) from mine tailing by employing Penicillium chrysogenum strain KBS3. The potential of Penicillium chrysogenum in generating a variety of organic acids was studied and the effect of different organic wastes as substrates was evaluated. Maximum solubilization of nickel (55%), copper (67%), magnesium (69%), cobalt (60%), and zinc (65%) was achieved in the media containing glucose, tea leaves, molasses, and waste winery grapes as substrates.

Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C

Using pulse electrophoresis in controlled homogenous electric field we conducted molecular karyotyping of highly-productive and laboratory strains of Acremonium chrysogenum generating antibiotic cephalosporin C (cefC). Differences in size of several chromosomes of highly active strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the highly active strain was not associated with alteration of localization and copy number ofcephalosporin C biosynthesis and transport genes. A cluster of "early" cefC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the "late genes", on chromosome II (2.3 Mb). Both clusters are presented as a single copy perA. chrysogenum genome in the wild-type and in CB26/8 producer strains. Based on comparative analysis of laboratory and industrial cefC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.

Preliminary bioleaching of heavy metals from contaminated soil employing indigenous Penicillium Chrysogenum strain F1

Bioleaching is an environment-friendly and economical technique to remove heavy metals from contaminated soil. The objective of this work is to find out an indigenous strain to remedy soil contaminated by Zn, Pb, Cu and Cd. A strain which was selected from the soil of a local smelting industry was found to be able to produce many organic acids and degrade pH value of the liquid medium. The fungus strain is identified as Penicillium Chrysogenum (P. Chrysogenum) by sequencing 18srDNA and ITS. Bioleaching condition using P. Chrysogenum is optimized. Glucose is the best carbon source for P. Chrysogenum and inorganic nitrogen is better than organic nitrogen. In addition, neutral solution and room temperature are fit for P. Chrysogenum to bioleach. In the one-step bioleaching, the bioleaching ratios are 39.95% for Zn, 9.4% for Pb, 34.89% for Cu and 49.59% for Cd, which are 53.89% for Zn, 14.44% for Pb, 55.53% for Cu and 62.81% for Cd in the two-step bioleaching. The efficiency of two-step bioleaching is better than the one-step bioleaching. P. Chrysogenum is effective in removing heavy metals from the contaminated soil.

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.

Degradation of 2,4-dichlorophenoxyacetic acid by a halotolerant strain of Penicillium chrysogenum: antibiotic production

The extensive use of pesticides in agriculture has prompted intensive research on chemical and biological methods in order to protect contamination of water and soil resources. In this paper the degradation of the pesticide 2,4-dichlorophenoxyacetic acid by a Penicillium chrysogenum strain previously isolated from a salt mine was studied in batch cultures. Co-degradation of 2,4-dichlorophenoxyacetic acid with additives such as sugar and intermediates of pesticide metabolism was also investigated. Penicillium chrysogenum in solid medium was able to grow at concentrations up to 1000 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) with sucrose. Meanwhile, supplementation of the solid medium with glucose and lactose led to fungal growth at concentrations up to 500 mg/L of herbicide. Batch cultures of 2,4-D at 100 mg/L were developed under aerobic conditions with the addition of glucose, lactose and sucrose, showing sucrose as the best additional carbon source. The 2,4-D removal was quantified by liquid chromatography. The fungus was able to use 2,4-D as the sole carbon and energy source under 0%, 2% and 5.9% NaCl. The greatest 2,4-D degradation efficiency was found using α -ketoglutarate and ascorbic acid as co-substrates under 2% NaCl at pH 7. Penicillin production was evaluated in submerged cultures by bioassay, and higher amounts of β -lactam antibiotic were produced when the herbicide was alone. Taking into account the ability of P. chrysogenum CLONA2 to degrade aromatic compounds, this strain could be an interesting tool for 2,4-D herbicide remediation in saline environments.

Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach

BackgroundIn microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited chemostat cultivations.ResultsDuring these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production.ConclusionsOur findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.

A preparative method for the purification of isopenicillin N from genetically blocked Acremonium chrysogenum strain TD189: studies on the degradation kinetics and storage conditions

A protocol for preparative isopenicillin N (IPN) purification, a highly interesting and hitherto unavailable intermediate of the penicillin and cephalosporin biosynthetic pathway due to its high unstability, is described. Culture broths of Acremonium chrysogenum TD189, a strain blocked in cephalosporin biosynthesis that accumulates this metabolite, were treated with acetone and filtered though charcoal and a hydrophobic resin in a single step as tandem columns. The cleared broth was then lyophilized and passed though a Sephadex G-25 column. The last step was the purification to homogeneity of IPN in a semipreparative HPLC equipment and, optionally, a desalting step by Sephadex G-10 column. Once purified, a complete analysis of the stability of the compound and the conditions for its long-term storage was carried out. Our results suggest a first-order model for IPN decomposition for all the pH and temperature analyzed. IPN is more stable at neutral pH, and once lyophilized, can be stored under vacuum and -75 ° C with a half-life of 770 days.

Phylogenetic Identification of Fungi Isolated from the Marine Sponge Tethya aurantium and Identification of Their Secondary Metabolites

Fungi associated with the marine sponge Tethya aurantium were isolated and identified by morphological criteria and phylogenetic analyses based on internal transcribed spacer (ITS) regions. They were evaluated with regard to their secondary metabolite profiles. Among the 81 isolates which were characterized, members of 21 genera were identified. Some genera like Acremonium, Aspergillus, Fusarium, Penicillium, Phoma, and Trichoderma are quite common, but we also isolated strains belonging to genera like Botryosphaeria, Epicoccum, Parasphaeosphaeria, and Tritirachium which have rarely been reported from sponges. Members affiliated to the genera Bartalinia and Volutella as well as to a presumably new Phoma species were first isolated from a sponge in this study. On the basis of their classification, strains were selected for analysis of their ability to produce natural products. In addition to a number of known compounds, several new natural products were identified. The scopularides and sorbifuranones have been described elsewhere. We have isolated four additional substances which have not been described so far. The new metabolite cillifuranone (1) was isolated from Penicillium chrysogenum strain LF066. The structure of cillifuranone (1) was elucidated based on 1D and 2D NMR analysis and turned out to be a previously postulated intermediate in sorbifuranone biosynthesis. Only minor antibiotic bioactivities of this compound were found so far.

The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White Biotechnology

The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54-1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54-1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.