Identification and Functional Analysis of the Mycophenolic Acid Gene Cluster of Penicillium roqueforti.

The filamentous fungus Penicillium roqueforti is widely known as the ripening agent of blue-veined cheeses. Additionally, this fungus is able to produce several secondary metabolites, including the meroterpenoid compound mycophenolic acid (MPA). Cheeses ripened with P. roqueforti are usually contaminated with MPA. On the other hand, MPA is a commercially valuable immunosuppressant. However, to date the molecular basis of the production of MPA by P. roqueforti is still unknown. Using a bioinformatic approach, we have identified a genomic region of approximately 24.4 kbp containing a seven-gene cluster that may be involved in the MPA biosynthesis in P. roqueforti. Gene silencing of each of these seven genes (named mpaA, mpaB, mpaC, mpaDE, mpaF, mpaG and mpaH) resulted in dramatic reductions in MPA production, confirming that all of these genes are involved in the biosynthesis of the compound. Interestingly, the mpaF gene, originally described in P. brevicompactum as a MPA self-resistance gene, also exerts the same function in P. roqueforti, suggesting that this gene has a dual function in MPA metabolism. The knowledge of the biosynthetic pathway of MPA in P. roqueforti will be important for the future control of MPA contamination in cheeses and the improvement of MPA production for commercial purposes.

Influence of intraspecific variability and abiotic factors on mycotoxin production in Penicillium roqueforti.

Penicillium roqueforti has the ability to produce secondary metabolites, including roquefortine C (ROQC) and mycophenolic acid (MPA). In a previous study, the presence of these mycotoxins, alone or in co-occurrence, has been reported in blue-veined cheese. A high variability of mycotoxin content has also been observed, although the majority of samples exhibited relatively low concentrations. The observed variability raises the question of the factors impacting ROQC and MPA production. In this context, the mycotoxigenic potential of 96 P. roqueforti strains (biotic factor) and the effect of some abiotic factors (pH, temperature, NaCl and O2 contents, and C/N ratio) on mycotoxin production were evaluated. A high intraspecific diversity, established via genotypic (RAPD) and phenotypic (FTIR) approaches, was observed. It was associated with mycotoxigenic potential variability and may thus explain part of the observed variability in mycotoxin content of blue-veined cheese. Moreover, a significant decrease of ROQC and MPA production was observed for conditions (temperature, C/N ratio, O2 and NaCl concentrations) encountered during cheese-making compared with optimal growth conditions. The results also highlighted that there was no significant effect of addition of ROQC amino-acid precursor on the production of both mycotoxins whereas a pH increase from 4.5 to 6.5 slightly reduced MPA but not ROQC production.

Immobilization technique for enhanced production of the immunosuppressant mycophenolic acid by ultraviolet and gamma-irradiated Penicillium roqueforti.

AIMS: Different entrapment matrices were screened to immobilize two strains of Penicillium roqueforti (AG101 and LG109) for more effective production of mycophenolic acid (MPA). Further improvement in the MPA productivity from immobilization of spores and mycelia was adopted by UV and gamma irradiation. METHODS AND RESULTS: Penicillium roqueforti strains were immobilized in different entrapping carriers and used for MPA production in shake flask cultures. Maximum MPA production was achieved on using an alginate concentration of 3·0% (w/v) and a mycelial fresh weight of 10% (w/v). MPA produced by alginate-immobilized spores and mycelia was almost double in comparison to the free system. The MPA-producing ability of immobilized AG101 and LG109 strain was significantly enhanced by mutagenesis through irradiation by UV (254 nm) for 120 and 90 min, respectively and gamma rays at 0·75 KGy. The feasibility of MPA production in a semi-continuous form by immobilized cells as affected by irradiation was adopted. CONCLUSIONS: MPA production by immobilized spores and mycelia was more intensified by UV and gamma irradiation. Moreover, the immobilized cell culture was superior to free-cell culture. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the future possibility to reduce the cost of producing fermentation-based drugs.

Optimization of submerged fermentation conditions for immunosuppressant mycophenolic acid production by Penicillium roqueforti isolated from blue-molded cheeses: enhanced production by ultraviolet and gamma irradiation.

Mycophenolic acid (MPA) is a promising drug owing to its immunosuppressive and biological activities. In this study, two strains of Penicillium roqueforti designated as AG101 and LG109 were selected among several strains isolated from Roquefort cheese samples on the basis of their activity for MPA-producing ability. The appropriate fermentation conditions necessary for MPA biosynthesis by the two respective fungal strains were investigated. These conditions included selection of the cultivation medium, agitation rate, incubation temperature, fermentation time, pH value, inoculum size, and fermentation medium volume. Maximum MPA productivities were maintained when the fermentation process was carried out using a medium composed of (g l(-1)): Sucrose, 30; peptone, 5.0; KH2PO4, 1.0; MgSO4·7H2O, 0.5 and KCl, 0.5; pH 6.0, inoculated with an inoculum size of 6.0 % (v/v), and incubated at 25 °C for 10 days at 120 rpm. The potentiality of both P. roqueforti strains for further improvement of MPA production was applied by mutagenesis through exposure to irradiation by ultraviolet rays (UV, 254 nm) for different periods of time and gamma rays at various doses (KGy). The dry cell weight of both irradiated fungal strains showed a greater reduction when irradiated either with UV or gamma rays. However, the MPA yield of both strains was increased by 1.27-1.39 fold when irradiated with UV rays and by 2.11-2.33 fold when irradiated with gamma rays, as compared with the respective controls (non-irradiated cultures). These findings indicate the future possibility to reduce the cost of producing fermentation-based drugs.

Production of 5-hydroxy-7-methoxy-4-methylphthalide in a culture of Penicillium crustosum.

The chemical reactions carried out by microorganisms have been used as a tool in modern chemistry. This paper reports the production of mycophenolic acid and a new phthalide by the endophytic fungus Penicillium crustosum obtained from coffee seeds. The fungus was cultivated in a liquid medium for a period of seven days and after that the culture medium was divided into four treatments: A, B, C and D, to which different organic substances were added. Treatment A was maintained as the control to evaluate the occurrence of biotransformation. Organic acids were added to the culture media of treatments B (ferulic and quinic acids) and C [cinnamic and 3,4-(methylenedioxy) cinnamic acids], and caffeine was added in the treatment D. All these organic compounds were dissolved in DMSO, and the fermentation was maintained for more 13 days, totalizing 20 days. Mycophenolic acid was isolated from the culture with no added acids (treatment A). Mycophenolic acid and a new phthalide, 5-hydroxy-7-methoxy-4-methylphthalide were isolated from treatments B and C, and mycophenolic acid and caffeine (added to the culture medium) were isolated from treatment D. The structures were determined by NMR techniques and confirmed by MS and MS/MS techniques.

[Establishment of a genetic transformation system for Penicillium brevicompactum to produce mycophenolic acid].

OBJECTIVE: We established a genetic transformation system for Penicillium brevicompactum to produce mycophenolic acid. METHODS: We developed protoplast transformation methods mediated by Polyethylene glycol, using phleomycin resistance gene (Sh ble) as a dominant selection marker. RESULT: The frequency of transformation was up to 2 - 3 transformants per microg DNA; analysis of the transformants by PCR showed that the foreign DNA had been integrated into the host genome. The transformants retained stable after generation. CONCLUSION: The establishment of the genetic transformation system of Penicillium brevicompactum could serve as the basis for the research of molecular biology and the breeding of gene engineering of the fungus.

Applications of ultrasound to enhance mycophenolic acid production.

Reducing production costs for fermentation-based drugs (e.g., antibiotics) is crucial for the long-term sustainability of healthcare. In this study, we propose a novel low-intensity pulsed ultrasound (LIPUS) stimulation scheme using a nominal frequency of 1.5 MHz with a 20% duty cycle (200 µs ultrasound on and 800 µs ultrasound off) to increase production of fermentation-based drugs. We chose Penicillium brevicompactum as a model system to demonstrate the performance of our LIPUS system. Penicillium brevicompactum can produce mycophenolic acid (MPA), an immunosuppressive agent commonly used to prevent rejection after organ transplantation. We have stimulated Penicillium brevicompactum in 50 mL shake flasks using LIPUS during its fermentation. After a series of screening experiments to optimize ultrasound parameters (e.g., ultrasound intensities, treatment duration and treatment frequency per day), it was concluded that ultrasound stimulation can increase MPA production by as much as 60% when treated eight times a day for 10-min durations at an intensity (spatial peak temporal average) of 200 mW/cm(2). These findings elucidate a new approach to reduce the cost of producing fermentation-based drugs.

Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis.

Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.

A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi.

BACKGROUND: Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF. RESULTS: In accordance with our hypothesis, heterologous expression of mpaF dramatically increased MPA resistance in a model fungus, Aspergillus nidulans, which does not produce MPA. The growth of an A. nidulans strain expressing mpaF was only marginally affected by MPA at concentrations as high as 200 µg/ml. To further substantiate the role of mpaF in MPA resistance, we searched for mpaF orthologs in six MPA producer/non-producer strains from Penicillium subgenus Penicillium. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting mpaF homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class. CONCLUSIONS: We identified a novel variant of the IMPDH-encoding gene in six different strains from Penicillium subgenus Penicillium. The novel IMPDH variant from MPA producer P. brevicompactum was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.

Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum.

Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.

[Producers of mycophenolic acid in ensiled and grain feeds].

Using the reaction of activated N-hydrooxisuccinimide ester of mycophenolic acid, a series of immunoreactive conjugated antigens with albumins, gelatin, and glucosoxidase is obtained. On the basis of polyclonal rabbit antibodies, a test-system for indirect competitive immunoenzyme analysis is elaborated, which has the sensitivity 0.4 ng/ml. By immunoanalysis, the ability for active biosynthesis of mycophenolic acid in strains of Byssochlamys nivea (44/44, 4100-68400 ng/ml) and Penicillium roqueforti (7/16, 204-25120 ng/ml) from the mycobiota of ensiled feeds is confirmed. The correspondence between weakly expressed producing capacity of most species of fungi of the genera Penicillium and Aspergillus prevailing in grain feeds and the data on low occurrence of this metabolite in grain (8.0%) and combined feeds (11.9%) is confirmed. A potential relationship between particular cases of a significant accumulation of mycophenolic acid (from 500 to 1500 microg/kg) in grains of wheat, corn, and combined feeds and a high biosynthetic activity in rare species P. puberulum, P. stoloniferum, and P. gladioli is discussed.

Growth and metabolites production by Penicillium brevicompactum in yoghurt.

Penicillium brevicompactum, commonly encountered in the indoor air, is known to produce a mycotoxin, mycophenolic acid (MPA). This mould has been isolated from a wide range of foods; considering that we had previously isolated this species from contaminated yoghurt, in this study we have evaluated its growth in yoghurt sweetened with sucrose, fructose and fructose added with fruit pieces. Fungal growth was evaluated monitoring CO(2) production in the headspace during yoghurt storage at 4+/-1, 8+/-1 and 10+/-1 degrees C throughout 21 days. P. brevicompactum grew well in the samples sweetened with fructose at 8 and 10 degrees C. The addition of sucrose influenced the growth negatively, particularly at 4 degrees C. Volatile Organic Compounds (VOC) and MPA production was determined at 8 degrees C in inoculated and uninoculated yoghurt, as well as in liquid malt extract. Differences in VOC profiles and in MPA production were correlated with the age of the fungus and with the growth medium. This study points out for the first time the early qualitative changes in volatile production patterns of a common indoor mould, grown in yoghurt, as well as the production of MPA during storage at refrigeration temperatures.

Determination of mycophenolic acid in meat products using mixed mode reversed phase-anion exchange clean-up and liquid chromatography-high-resolution mass spectrometry.

A method for determination of mycophenolic acid (MPA) in dry-cured ham, fermented sausage and liver pâté is described. MPA was extracted from meat with bicarbonate-acetonitrile, further cleaned-up by mixed mode reversed phase-anion exchange and detected using a LC-MS system with electrospray ionisation-time-of-flight detection. The limit of detection was 4 microg/kg in sausage and 6 microg/kg in ham and pâté. The method was successfully used for quantification of MPA in dry-cured ham and liver pâté artificially inoculated with Penicillium brevicompactum. Levels ranged from 190 microg/kg in centre to 11 mg/kg in surface of ham and from 150 microg/kg in bottom to 14 mg/kg in surface of pâté.

Polymerase chain reaction (PCR) identification of Penicillium brevicompactum, a grape contaminant and mycophenolic acid producer.

Penicillium brevicompactum is a ubiquitous fungal species that contaminates diverse substrates and commodities and produces an array of metabolites toxic to human and animals. The present work has obtained evidence, by liquid chromatography (LC)-ion-trap mass spectrometry, of the ability of P. brevicompactum strains isolated from grapes to produce mycophenolic acid, a potent immunosuppressor. In order to facilitate early diagnosis of this species on commodities for human and animal consumption, a rapid, sensitive and specific polymerase chain reaction (PCR) assay for P. brevicompactum was developed. The specific primers were designed based on the ITS1-5.8S-ITS2ITS (Internal Transcribed Spacers of rRNA genes) multicopy region. This method provides a useful aid to detect the presence of this fungal species in grapes and other commodities in order to prevent the toxins produced entering the food chain.

[Production of mycophenolic acid by fungi of the genus Penicillium link].

Out of 36 strains of fungi of the genus Penicillium, some of which were isolated from ancient permafrost soils, 14 strains synthesized mycophenolic acid (MPA). Maximal (over 500 mg/l) accumulation of MPA in culture liquid was observed in P. brevicompactum strains (VKM F-457, VKM F-477, and VKM F-1150). This was the first study to detect MPA in representatives of the species P. rugulosum; in three strains of this species (VKM FW-665, VKM FW-717, and VKM FW-733), the level of MPA accumulation exceeded 300 mg/l. The time course of the synthesis of MPA by the P. rugulosum strain VKM FW-733 was studied. It was shown that the synthesis of this metabolite was dramatically intensified at the stationary growth phase (ten days).

Byssochlamys nivea as a source of mycophenolic acid.

Byssochlamys species are responsible for spoilage and degradation of fruits and silages and can also produce the mycotoxin patulin. We analyzed secondary metabolite production by Byssochlamys nivea. Mycophenolic acid and its precursors, 5-methylorsellinic acid and 5,7-dihydroxy-4-methylphthalide, were identified in all of the B. nivea strains that we examined.

Pharmacokinetics of tacrolimus and mycophenolic acid are altered, but recover at different times during hepatic regeneration in rats.

Hepatic regeneration is very critical to the success of living donor liver transplantation, which allows a reduced size liver to grow in size to accommodate the requirements of both the donor and the recipient. The objectives of this study were to evaluate 1) the hepatic metabolism of the two immunosuppressive drugs, tacrolimus and mycophenolic acid (MPA), and 2) the pharmacokinetics of tacrolimus and mycophenolic acid at various time points after initiation of hepatic regeneration by partial hepatectomy in rats. The hepatic intrinsic clearance of tacrolimus was decreased to 70% and 51% of the control level at the 24th h and the 6th day, respectively, but returned to normal level by day 14. The total body clearance of tacrolimus was reduced transiently but recovered completely by day 18. The hepatic intrinsic clearance of MPA was decreased to 52% and 51% of that in control rats at the 24th h and the 6th day, respectively, but recovered to normal level by day 14. The total body clearance of MPA was reduced at the 24th h but recovered by day 6. The magnitude of reduction in the clearance of tacrolimus and MPA was much smaller than what was predicted from in vitro data. The elimination clearance of MPA glucuronide was also impaired during hepatic regeneration but recovered to normal level with time. In conclusion, the pharmacokinetics of tacrolimus and mycophenolic acid were altered during hepatic regeneration but recovered completely at different rates over time. Caution must be exercised in extrapolating in vitro data to in vivo conditions during hepatic regeneration.

Radiogas chromatography mass spectrometry in the selected ion monitoring mode.

The value of selected ion monitoring in analyzing biological radio isotope incorporation experiments by radiogas chromatography mass spectrometry is illustrated with reference to the biosynthesis of the mycotoxin mycophenolic acid and the mode of action of the anticholesterolemic drug 20,25-diazacholesterol. It is shown that the increased sensitivity and specificity of the selected ion monitoring mode detector permits straightforward detection and identification of the relatively small cellular pools associated with metabolic intermediates. The computer program RADSIM is described. Problems that still exist in using radiogas gas chromatography mass spectrometry technology to analyse isotope incorporation experiments are discussed.

Mycophenolic acid production by Penicillium brevicompactum in two media.

Penicillium brevicompactum produces mycophenolic acid as it grows vegetatively, not only on a simple medium where growth is slow but also on a richer medium where growth is less restricted. The implications of this finding on the association of fungal secondary metabolism with the idiophase in liquid and solid culture are discussed.