Penicillium Chrysogenum Strain Research Articles

Medicinal Polypores of the Forests of North America: Screening for Novel Antiviral Activity

362 International Journal of Medicinal Mushrooms Polypore mushrooms have been used medicinally for thousands of years. Th e Greek physician Dioscorides fi rst described the use of a wood conk, Agarikon, now known as Fomitopsis offi cinalis (Vill.: Fr.) Bond. et Singer (= Laricifomes offi cinalis), as a treatment against consumption in 65 AD. Other wood conks, such as Ling Chi or Reishi, have had a similarly long history of use in Asia. In the past 20 years, wood conks have been carefully explored for their immunomodulating and anticancer properties. More recently, mushrooms, including polypores, have and are being explored for their antimicrobial properties. Upon submitting more than a hundred in vitro cultures of mushrooms to the US Defense Department’s Bioshield BioDefense program, several tests show that some of these polypore mushrooms have strong antiviral activity. Within these verdant natural landscapes, trees hundreds of years old host ancestral strains of these elusive polypores. Species that are now rare, or in some cases thought to be extinct, still reside in the pristine old growth forests of the Pacifi c Northwest of North America. When clones from these mushrooms were grown in vitro and submitted for antiviral screening, several mycelial cultures produced antibiotics eff ective against Pox and other viruses. Notably, strains vary in their antiviral properties. Our natural genomes hold within them great potentials for staving off disease and have not yet been fully explored. Th e fungal diversity within these genomes may prove critical for isolating the most active strains, similar to the lessons learned from the isolation of Penicillium chrysogenum strains that lead to the commercialization of penicillin and saved millions of lives. With deforestation, pollution, and industrialization, societies should reevaluate the importance of their natural forests in the context that they hold within them novel medicines of enormous socioeconomic importance. Th e old paradigm of viewing the forest as valuable only in terms of timber seems overly simplistic given this new knowledge. Medicinal Polypores of the Forests of North America: Screening for Novel Antiviral Activity

Medicinal Polypores of the Forests of North America: Screening for Novel Antiviral Activity

Polypore mushrooms have been used medicinally for thousands of years. The Greek physician Dioscorides first described the use of a wood conk, Agarikon, now known as Fomitopsis officinalis (Vill.: Fr.) Bond. et Singer (= Laricifomes officinalis), as a treatment against consumption in 65 AD. Other wood conks, such as Ling Chi or Reishi, have had a similarly long history of use in Asia. In the past 20 years, wood conks have been carefully explored for their immunomodulating and anticancer properties. More recently, mushrooms, including polypores, have and are being explored for their antimicrobial properties.Upon submitting more than a hundred in vitro cultures of mushrooms to the US Defense Department’s Bioshield BioDefense program, several tests show that some of these polypore mushrooms have strong antiviral activity. Within these verdant natural landscapes, trees hundreds of years old host ancestral strains of these elusive polypores. Species that are now rare, or in some cases thought to be extinct, still reside in the pristine old growth forests of the Pacific Northwest of North America. When clones from these mushrooms were grown in vitro and submitted for antiviral screening, several mycelial cultures produced antibiotics effective against Pox and other viruses. Notably, strains vary in their antiviral properties. Our natural genomes hold within them great potentials for staving off disease and have not yet been fully explored. The fungal diversity within these genomes may prove critical for isolating the most active strains, similar to the lessons learned from the isolation of Penicillium chrysogenum strains that lead to the commercialization of penicillin and saved millions of lives.With deforestation, pollution, and industrialization, societies should reevaluate the importance of their natural forests in the context that they hold within them novel medicines of enormous socio-economic importance. The old paradigm of viewing the forest as valuable only in terms of timber seems overly simplistic given this new knowledge.

Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Kinetics of adipoyl-7-aminodeacetoxycephalosporanic acid and byproduct formations.

The production kinetics of a transformed strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was investigated in chemostat cultivations. The recombinant strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) as the major product; however, during the cultivations, the appearance of a major unknown and poorly secreted product was observed. Investigations using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS) showed that this byproduct has a six-membered dihydrothiazine ring, which is characteristic for cephalosporins. The byproduct may be formed via isopenicillin N by as-yet unknown mechanisms, but involving expandase. It is likely that the unknown compound (UC) is deacetoxycephalosporin C (DAOC). Investigation of the instability of the various beta-lactams produced showed higher instability for compounds with a five-membered thiazolidine ring than those with a six-membered dihydrothiazine ring. Furthermore, secretion of products and byproducts was shown to be quite different. The productivity was studied as a function of the dilution rate in the range 0.015 to 0.090 h(-1). The specific productivity of total beta-lactams was compared with that of the penicillin-G-producing host strain, and it was found to be lower at dilution rates of <0.06 h(-1). Quantification of the fluxes through the pathway leading to ad-7-ADCA showed a decrease in flux toward ad-7-ADCA, and an increase in flux toward UC as the dilution rate increased. Northern analysis of the biosynthetic genes showed that expression of the enzymes involved in the ad-7-ADCA pathway decreased as the dilution rate increased.

Influence of the adipate and dissolved oxygen concentrations on the β-lactam production during continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus

The influence of adipate concentration and dissolved oxygen on production of adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) by a recombinant strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was studied in glucose-limited continuous cultures. Operating conditions were maintained constant but the adipate and dissolved oxygen concentrations (DOC) were varied separately in a range from 1 to 37.5 g l −1 and from 2% to 125% air saturation (%AS), respectively. The total β-lactams specific productivity, r p total , was not significantly changed for adipate concentrations from 5 to 25 g l −1 , but the flux towards an unknown by-product decreased as the adipate concentration increased. Investigations at different DOC showed that r p total was stable around 18 μmol g DW −1 h −1 for DOC being in the range from 15 to 125%AS. When DOC was decreased from 15 to 7%AS, r p total increased to 25 μmol g DW −1 h −1 , mainly due to a two-fold increase in the adipoyl-6-aminopenicillanic acid (ad-6-APA) specific productivity.

Nitrate regulation of alpha-aminoadipate reductase formation and lysine inhibition of its activity in Penicillium chrysogenum and Acremonium chrysogenum.

alpha-Aminoadipate reductase (alpha-AAR) is a key enzyme in the branched pathway for lysine and beta-lactam biosynthesis of filamentous fungi since it competes with alpha-aminoadipyl-cysteinyl-valine synthetase for their common substrate L-alpha-aminoadipic acid. The alpha-AAR activity in two penicillin-producing Penicillium chrysogenum strains and two cephalosporin-producing Acremonium chrysogenum strains has been studied. The alpha-AAR activity peaked during the growth-phase preceding the onset of antibiotic production, which coincides with a decrease in alpha-AAR activity, and was lower in high penicillin- or cephalosporin-producing strains. The alpha-AAR required NADPH for enzyme activity and could not use NADH as electron donor for reduction of the alpha-aminoadipate substrate. The alpha-AAR protein of P. chrysogenum was detected by Western blotting using anti-alpha-AAR antibodies. The mechanism of lysine feedback regulation in these two filamentous fungi involves inhibition of the alpha-AAR activity but not repression of its synthesis by lysine. This is different from the situation in yeasts where lysine feedback inhibits and represses alpha-AAR. Nitrate has a strong negative effect on alpha-AAR formation as shown by immunoblotting studies of alpha-AAR. The nitrate effect was reversed by lysine.

The biosynthesis of low-molecular-weight nitrogen-containing secondary metabolite-alkaloids by the resident strains of Penicillium chrysogenum and Penicillium expansum isolated on the board of the Mir space station

The analysis of the absorption spectra of the low-molecular-weight nitrogen-containing secondary metabolites--alkaloids--of 4 Penicillium chrysogenum strains and 6 Penicillium expansum strains isolated on board the Mir space station showed that all these strains synthesize metabolites of alkaloid origin (roquefortine, 3,12-dihydroroquefortine, meleagrin, viridicatin, viridicatol, isorugulosuvin, rugulosuvin B, N-acetyl-tryptamine, and a "yellow metabolite" containing the benzoquinone chromophore).

Metabolism of the phosphonate herbicide glyphosate by a non-nitrate-utilizing strain of Penicillium chrysogenum.

A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containing the herbicide glyphosate as the only nitrogen source. The presence of concentrations up to 25 mM progressively stimulated the fungal growth rate, which was negligible in media lacking reduced nitrogen. However, glyphosate utilization never exceeded 1 mmol g-1 mycelial dry mass, and below a threshold concentration both herbicide uptake and fungal growth were subject to a lag phase, suggesting that the herbicide may enter the cell by either simple passive diffusion or inducible carriers. Amino acids, possible products of glyphosate breakdown, as well as ammonia, were found to replace the herbicide in restoring mycelial growth. Cells were devoid of detectable nitrate reductase activity, thus the isolate seems to be impaired in its ability to convert nitrate to ammonium. In vitro activity of 5-enol-pyruvyl-shikimate-3-phosphate synthase, the target site of glyphosate action, was highly sensitive to the herbicide. Fungal growth rate was considerably lower when the herbicide was also the only phosphorus source, whereas glyphosate utilization was substantially unaffected, suggesting an unusual route for its degradation. Herbicide metabolism was strongly reduced when other sources of organic nitrogen were made available.

Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum.

A recently developed method for analyzing metabolic networks using 13C-labels was employed for investigating the metabolism of a high- and a low-yielding strain of Penicillium chrysogenum. Under penicillin-producing conditions, the flux through the pentose phosphate (PP) pathway in the high- and the low-yielding strains was estimated to 70 and 66, respectively. When the high-yielding strain was cultivated in a medium without the penicillin side chain precursor, phenoxyacetic acid, the PP pathway flux was estimated as 71. Thus, in all three experiments, the flux through the PP pathway was almost constant with an average value of 69+/-3, and the method therefore allows for a very reproducible estimation of the PP pathway flux. Phenoxyacetic acid was found to be a source of cytosolic acetyl-CoA and thereby a source of precursors for the biosynthesis of 2-aminoadipic acid, which is a central amino acid in penicillin biosynthesis. However, the labeling patterns also indicated the presence of an unrecognized pathway to cytosolic acetyl-CoA.

Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum.

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.

Molecular tools for identification of Penicillium starter cultures used in the food industry

The main goal of this work was to develop rapid and accurate molecular tools to discriminate species of white industrial Penicillia. We applied three different polymerase chain reaction (PCR) based techniques. Sequences of the ITS region of the rRNA gene unit and of the 5′ end of the β tubulin gene yielded 1.2% and 5.8% nucleotide variability respectively, between Penicillium camembertii and Penicillium nalgiovense. Polymorphic restriction sites were found in both sequences. These may be used in diagnostic PCR–RFLP analysis to rapidly distinguish between the two Penicillium species. Random amplified polymorphic DNA (RAPD) markers were also useful to differentiate these two species, but no polymorphism was found at the subspecific level, which evidenced a high level of homogeneity of the isolates studied. By means of these three techniques, the real identity of industrial strains of Penicillium chrysogenum and P. nalgiovense could be demonstrated. The comparison of these isolates with type strains of the two species suggested that the former corresponds to P. nalgiovense. The genetic relatedness between P. naglovense and Penicillium dipodomyis was also confirmed.

Disruption of phacA, an Aspergillus nidulans Gene Encoding a Novel Cytochrome P450 Monooxygenase Catalyzing Phenylacetate 2-Hydroxylation, Results in Penicillin Overproduction

Aspergillus nidulans utilizes phenylacetate as a carbon source via homogentisate, which is degraded to fumarate and acetoacetate. Mutational evidence strongly suggested that phenylacetate is converted to homogentisate through two sequential hydroxylating reactions in positions 2 and 5 of the aromatic ring. Using cDNA substraction techniques, we have characterized a gene, denoted phacA, whose transcription is strongly induced by phenylacetate and which putatively encodes a cytochrome P450 protein. A disrupted phacA strain does not grow on phenylacetate but grows on 2-hydroxy- or 2, 5-dihydroxyphenylacetate. Microsomal extracts of the disrupted strain are deficient in the NADPH-dependent conversion of phenylacetate to 2-hydroxyphenylacetate. We conclude that PhacA catalyzes the ortho-hydroxylation of phenylacetate, the first step of A. nidulans phenylacetate catabolism. The involvement of a P450 enzyme in the ortho-hydroxylation of a monoaromatic compound has no precedent. In addition, PhacA shows substantial sequence divergence with known cytochromes P450 and defines a new family of these enzymes, suggesting that saprophytic fungi may represent a source of novel cytochromes P450. Phenylacetate is a precursor for benzylpenicillin production. phacA disruption increases penicillin production 3-5-fold, indicating that catabolism competes with antibiotic biosynthesis for phenylacetate and strongly suggesting strategies for Penicillium chrysogenum strain improvement by reverse genetics.

Uptake of phenylacetic acid by two strains of Penicillium chrysogenum.

Uptake of phenylacetic acid, the side-chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54-1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high-yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid-derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250-3500 microM), and in the high-yielding strain at low phenylacetic acid concentrations (2. 8-100 microM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC-analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]-labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport-assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high-affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54-1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin.

Analysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster.

Several commercially improved strains of Penicillium chrysogenum have been shown to carry amplifications of the entire penicillin biosynthesis gene cluster. Analysis previously carried out using the strain BW 1890 has here been extended to the characterisation of other members of the SmithKline Beecham strain improvement series. We have determined the length of the amplicon to be 57.4 kb and shown a general increase in copy number and penicillin titre through the series. Sequence analyses of the promoter regions of the acvA, ipnA and aat genes in the high titre strain BW 1901, and comparisons with wild-type sequences have not identified any potentially titre-enhancing mutations. In addition, cDNA screening has failed to identify any further transcribed elements within the co-amplified region. The homogeneity of hybridisation patterns and the identification and analysis of a single copy revertant has shown that the amplification is of a direct tandem nature and we propose a model of chromatid misalignment and recombination as its mode of generation. Hybridisation analysis of penicillin non-producing mutants has indicated the loss, in all those investigated, of the entire penicillin biosynthesis gene cluster, similarities between the deletion junctions in these strains and comparison with previously published data indicating the presence of recombinogenic regions flanking the penicillin biosynthesis gene cluster.

Molecular Cloning and Expression in Different Microbes of the DNA Encoding Pseudomonas putida U Phenylacetyl-CoA Ligase:: USE OF THIS GENE TO IMPROVE THE RATE OF BENZYLPENICILLIN BIOSYNTHESIS IN PENICILLIUM CHRYSOGENUM

The gene encoding phenylacetyl-CoA ligase (pcl), the first enzyme of the pathway involved in the aerobic catabolism of phenylacetic acid in Pseudomonas putida U, has been cloned, sequenced, and expressed in two different microbes. In both, the primary structure of the protein was studied, and after genetic manipulation, different recombinant proteins were analyzed. The pcl gene, which was isolated from P. putida U by mutagenesis with the transposon Tn5, encodes a 48-kDa protein corresponding to the phenylacetyl-CoA ligase previously purified by us (Martínez-Blanco, H., Reglero, A. Rodríguez-Aparicio, L. B., and Luengo, J. M. (1990) J. Biol. Chem. 265, 7084-7090). Expression of the pcl gene in Escherichia coli leads to the appearance of this enzymatic activity, and cloning and expression of a 10.5-kb DNA fragment containing this gene confer this bacterium with the ability to grow in chemically defined medium containing phenylacetic acid as the sole carbon source. The appearance of phenylacetyl-CoA ligase activity in all of the strains of the fungus Penicillium chrysogenum transformed with a construction bearing this gene was directly related to a significant increase in the quantities of benzylpenicillin accumulated in the broths (between 1.8- and 2.2-fold higher), indicating that expression of this bacterial gene (pcl) helps to increase the pool of a direct biosynthetic precursor, phenylacetyl-CoA. This report describes the sequence of a phenylacetyl-CoA ligase for the first time and provides direct evidence that the expression in P. chrysogenum of a heterologous protein (involved in the catabolism of a penicillin precursor) is a useful strategy for improving the biosynthetic machinery of this fungus.

Molecular genetics as a tool to remove bottlenecks in the biosynthesis of ?-lactam antibiotics

Several strains of Penicillium chrysogenum with different productions of penicillin were characterized at the molecular level in order to establish the basis of the increased penicillin production rates. The cluster of penicillin biosynthetic genes was located in an amplified genomic region of 57.9 kb in a high-producing strain (E1) and 106.5 kb in two strains (AS-P-78 and P2) producing moderately high levels of penicillin. This region was shown to be present in multiple tandemly repeated copies with a different copy number depending on the strain. The sequence TTTACA appeared at the junction points between repeats and at the borders of the amplified region in strains AS-P-78 and P2, while its reverse complementary TGTAAA was found in strain E1. The tandem reiteration and deletion appear to arise by site-specific recombination induced by mutagenic treatments. Finally, the relationship between glucose repression and pH regulation was studied in strain AS-P-78.

Composition and toxigenic potential of the mould population on dry-cured Iberian ham

The fungal population on dry-cured Iberian ham can be essential to the development of the product's unique characteristics, but health hazards due to mycotoxins may be significant. We examined the natural fungal population of Iberian hams during ripening at three different locations. Chloroform extracts from 59 selected isolates were tested for toxicity to brine shrimp larvae and VERO cells, for mutagenicity in the Ames test and for antimicrobial activity against Staphylococcus aureus. The diversity of moulds increased during ripening. Penicillium commune, Penicillium chrysogenum, Penicillium aurantiogriseum, Penicillium expansum and Penicillium echinulatum dominated most of the ripening time; however, the Eurotium species, particularly E. herbariorum and E. repens, increased in the final product. Using the above tests, most moulds were toxigenic. The toxigenic potential of the fungal population increased as the processing progressed. To minimize health hazards from uncontrolled fungal populations, we identified non toxigenic strains of Penicillium chrysogenum that could be used as starters in dry-cured hams.

BMS-182123, a fungal metabolite that inhibits the production of TNF-alpha by macrophages and monocytes.

A fungal metabolite, BMS-182123, which inhibited bacterial endotoxin-induced production of tumor necrosis factor (TNF-alpha) in murine macrophages and human peripheral blood monocytes (in vitro), was isolated from the culture broth of Penicillium chrysogenum strain V39673. The effective BMS-182123 concentration (IC50) resulting in 50% inhibition of lipopolysaccharide-induced TNF-alpha production in murine macrophages and human monocytes was 600 ng/ml and 4.0 microgram/ml, respectively. BMS-182123 suppressed the lipopolysaccharide-induced TNF-alpha promoter activity and did not affect the stability of posttranscriptional mRNA. Addition of hydrophobic resin, Amberlite XAD-8 (1%), to the fermentation enhanced the production of BMS-182123 by 5.5 fold. A total of 577 mg pure BMS-182123 was recovered from a 250-liter fermentation supplemented with 1% Amberlite XAD-8.

ChemInform Abstract: Isolation and Identification of a New Cephem Compound from Penicillium chrysogenum Strains Expressing Deacetoxycephalosporin C Synthase Activity

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Rapid and quantitative analysis of metabolites in fermentor broths using pyrolysis mass spectrometry with supervised learning: application to the screening of Penicillium chrysogenum fermentations for the overproduction of penicillins

The combination of pyrolysis mass spectrometry (PyMS) and artificial neural networks (ANNs) can be used to quantify levels of penicillins in strains of Penicillium chrysogenum and ampicillin in spiked samples of Escherichia coli. Four P. chrysogenum strains (NRRL 1951, Wis Q176, P1, and P2) were grown in submerged culture to produce penicillins, and fermentation samples were taken aseptically and subjected to PyMS. To deconvolute the pyrolysis mass spectra so as to obtain quantitative information on the titre of penicillins, fully-interconnected feedforward artificial neural networks (ANNs) were studied; the weights were modified using the standard back-propagation algorithm, and the nodes used a sigmoidal squashing function. In addition the multivariate linear regression techniques of partial least squares regression (PLS), principal components regression (PCR) and multiple linear regression (MLR) were applied. The ANNs could be trained to give excellent estimates for the penicillin titre, not only from the spectra that had been used to train the ANN but more importantly from previously unseen pyrolysis mass spectra. All the linear regression methods failed to give accurate predictions, because of the very variable biological backgrounds (the four different strains) in which penicillin was produced and also of the inability of models using linear regression accurately to map non-linearities. Comparisons of squashing functions on the output nodes of identical 150-8-1 neural networks revealed that networks employing linear functions gave more accurate estimates of ampicillin in E. coli near the edges of the concentration range than did those using sigmoidal functions. It was also shown that these neural networks could be successfully used to extrapolate beyond the concentration range on which they had been trained. PyMS with the multivariate clustering technique of principal components analysis was able to differentiate between four strains of P. chrysogenum studied, and was also able to detect phenotypic differences at five, seven, nine or 11 days growth. A crude smapling procedure consisting of homogenised agar plugs proved applicable for rapid analysis of a large number of samples.

Uptake of phenoxyacetic acid by Penicillium chrysogenum.

The uptake of phenoxyacetic acid by two different strains of Penicillium chrysogenum was studied. Phenoxyacetic acid (POA) was taken up by P. chrysogenum in a defined medium. Plots of initial velocity of POA uptake versus external substrate concentration, in the range 2-5000 microM, gave linear plots. Uptake of POA by induced and uninduced cells was identical. The initial velocity of POA uptake decreased as the pH of the suspension was increased from 5.4 to 7.2; the decrease closely paralleled the decline in the non-ionic form of the acid over this pH range. The initial velocity of POA uptake was not affected by the presence of phenylacetic acid. POA uptake proceeded until the cellular concentration was equal to the external concentration. It is concluded that POA is passively transported into P. chrysogenum by unmediated diffusion.