Production of a heterologous recombinant protein using fragments of the glyceraldehyde-3-phosphate dehydrogenase promoter from Penicillium camemberti

The biotechnological applications of cheese-ripening fungi have been limited by a lack of genetics tools, in particular the identification and characterization of suitable promoters for protein expression. In this study, the suitability of the glyceraldehyde-3-phosphate dehydrogenase (gpdP) promoter from Penicillium camemberti to drive the production of a recombinant protein was evaluated. The gpdP gene and its promoter were isolated using PCR and Genome Walker. The promoter of gpdP has two regions with high identity to the regulatory elements gpd-box and ct-box previously described in Aspergillus nidulans. Two fragments of the promoter containing the gpd- and ct-box or the ct-box alone were used to drive the in vivo production of recombinant β-galactosidase using A. nidulans as host. Our results indicate that larger fragment containing gpd-box enhances the production of β-galactosidase activity levels respect to ct-box alone, and that both boxes are necessary to obtain maximal enzymatic activity production. The smaller fragment (187 nt) containing the ct-box alone was able to trigger up to 27% of β-galactosidase activity, and to our knowledge this is the smallest fragment from a gpd gene used to produce a recombinant protein. Differences were not observed when glycerol, galactose or glucose were used as carbon sources, suggesting that the promoter activity is carbohydrate-independent. This is the first report in which a Penicillium gpd promoter is used for recombinant protein production. Our results open the way for the future development of a system for recombinant proteins expression in the biotechnologically important cheese-ripening fungus P. camemberti.     

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5′-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.     

Penicillium roqueforti: a multifunctional cell factory of high value‐added molecules

This is a comprehensive review, with 114 references, of the chemical diversity found in the fungus Penicillium roqueforti. Secondary metabolites of an alkaloidal nature are described, for example, ergot alkaloids such as festuclavine, isofumigaclavines A and B, and diketopiperazine alkaloids such as roquefortines A–D, which are derived from imidazole. Other metabolites are marcfortines A–C, PR‐toxin, eremofortines A–E, mycophenolic and penicillic acids, and some γ‐lactones. Also, recent developments related to the structural characteristics of botryodiplodin and andrastin are studied—the latter has anticancer properties. Finally, we discuss the enzymes of P. roqueforti, which can participate in the biotechnological production of high value‐added molecules, as well as the use of secondary metabolite profiles for taxonomic purposes.     

Isolation and metabolite production by Penicillium roqueforti,P. paneum and P. crustosum isolated in Canada

Penicillium roqueforti, P. crustosum and P. paneum grow on ensiled grain and recycled feed unless properly treated. The former two species occur also on cut lumber in Canada. These are known to produce a number of secondary metabolites including roquefortine. In cooler dairy production areas, including Scandinavia and North America, cattle toxicosis has been associated with silage contaminated by these fungi. We collected strains associated with cow or cattle toxicoses. The principal metabolites were determined making use of a new extraction method and analysis combining HPLC, LC/MS/MS, and LC/NMR. Penicillium roqueforti and P. crustosum required amino acid nitrogen for metabolite formation and their toxins were formed under conditions of low oxygen (20–30% saturation). Production of roquefortine C occurred on depletion of the available nitrogen and penitrem A on depletion of carbon source. Yield was reduced by excess carbon. Medium osmotic tension (a_w) affected metabolite production by the two species differently. Penicillium paneum was associated with ill-thrift of dairy cows and P. roqueforti was associated with more serious symptoms. Our data suggest a physiological basis for the common occurrence of roquefortine C in silage without serious consequences and the alternative, the presence of roquefortine C and toxicoses. The strain isolated from lumber was the best producer of the toxins studied. This is the first report of the toxigenic potential of P. roqueforti and P. paneum from Canada.     

Measurement of Mercury Methylation in Lake Water and Sediment Samples

The production of various eremophilane-type sesquiterpenes by Penicillium roqueforti strains has allowed us to propose a biochemical pathway for PR toxin synthesis. A time-course study of P. roqueforti metabolite production by high-performance liquid chromatography was performed to check this hypothetical pathway. The results obtained suggested that eremofortin C was the direct precursor of PR toxin in the P. roqueforti cell. Attempts to determine the amount of PR toxin in the mycelium failed. It was shown that the absence of PR toxin in mycelium was due to its instability during the extraction procedure.     

Aroma-Forming Substances from a Culture of the Mold Fungus Penicillium roquefortiGrown on Curd

Production of aroma-forming substances by Penicillium roquefortistrains No. 31 and No. 541-A grown on curd was studied. The data showed that strain No. 541-A is the most promising producer of cheese flavor. The flavor acquired a soily scent after more than 5 days cultivation, which may hamper the use of these cultures (particularly, No. 31) in the food industry.     

Toxigenic fungi isolated from roquefort cheese

To evaluate the potential for mycotoxin production by fungi contaminating blue-veined cheese, as well as by the ripening fungus,Penicillium roqueforti, the fungal flora of six of local and imported brands was determined. A total of 19 fungi were isolated from the six brands tested. Fourteen of the isolates were toxic to chicken embryos. The toxigenic fungi produced the following mycotoxins:Aspergillus fumigatus, kojic acid;A. versicolor, sterigmatocystin;Penicillium roqueforti, penicillic acid and unidentified toxic metabolites.     

Growth of Geotrichum candidum and Penicillium camemberti Cultivated on Liquid Media Correlated with Ammonia and Methanethiol Emission

The growth of Geotrichum candidum and Penicillium camemberti plays an important role in the ripening of Camembert‐type cheeses, but the monitoring of the corresponding kinetics for fungal cocultures on solid media appears difficult. Continuous and non‐intrusive methods to characterize the growth of both species (like the monitoring of the emissions of ammonia and volatile sulphur compounds) may be highly relevant, under the condition that such emissions could be correlated with growth. This would be easier to investigate in submerged culture, since total biomass concentration is known to vary in proportion to broth turbidity. For this reason, growth kinetics, ammonia and flavour gas emission of both Geotrichum candidum and Penicillium camemberti grown separately in submerged cultures under the conditions of low aeration rate and uncontrolled pH were continuously recorded.In the basal medium (peptone+lactate supplemented with both glutamic acid and methionine [1 g/l]each), no significant gas emission was observed during the growth of both fungi. Ammonia and sulphur gas emissions by G. candidum were a little stimulated by supplementing the basal medium with trace elements, and, at a larger extent, by the addition of inorganic phosphate: Such a gaseous emission took place at the end of the growth phase of G. candidum. Irrespective of the basal medium supplementation, no significant emission ofammonia and sulphur gas was observed during the growth of P. camemberti. For the media and strains used, ammonia and volatile sulphur compounds emissions unequivocally showed the growth of Geotrichum candidum.     

Studies on the PR toxin of penicillium roqueforti

A mycotoxin, confirmed by chemical, physical and spectroscopic data as the PR toxin described by Ru-Dong Wei and coll. (15) has been isolated from culture filtrates of Penicillium roqueforti Thom. Factors affecting the toxin and mycelium production, acute and chronic toxicity in experimental animals and the frequency of toxinogenesis of 21 isolates of P. roqueforti (including a brown mutant) isolated from different materials, foods especially, were also studied. An hypothesis on the absence of PR toxin in cheeses fermented with P. roqueforti is also advanced.     

Comparison of growth characteristics and roquefortin C production of<Emphasis Type="Italic">Penicillium roqueforti</Emphasis> from blue-veined cheese

The properties of 21 isolates ofPenicillium roqueforti from just as many commercial blue-veined cheeses, purchased from the Argentinean market (domestic and imported products) were comparatively examined. Isolates were investigated for their ability to grow at different temperatures, pH values and concentration of NaCl, as well as for their proteolytic and lipolytic activities, respectively. The potential of these strains to produce roquefortin in vitro, and the actual levels of roquefortin in 10 of these cheeses were analysed by TLC. All strains showed similar growth properties in aspects of salt concentration and pH-value of the medium, and all grew well at 10 °C. Only four strains showed proteolytic activity on casein agar, while all strains were lipolytic on trybutirin agar. After incubation at 25 °C for 16 days, all strains produced roquefortin in Yeast Extract Sucrose (25.6–426.7 μg/g) and in reconstituted (10%) sterile skim milk (26.9–488 μg/g). Roquefortin at >0.1 μg/g was also found in 9 out of 10 analysed samples of blue-veined cheeses (8 from Argentine, 1 from Spain), with a maximum value 3.6 μg/g. During the ripening process of blueveined cheese, production of roquefortin seems to be unavoidable. Care should be taken to select strains with low toxin production characteristics, to minimize potential health risks. Roquefortin C production byP. roqueforti in vitro was not correlated with roquefortin C levels found in cheese. Financial support: Research grants from the National University of Quilmes, Argentina     

Simple aid for the identification of Penicillium roqueforti Thom

Summary 53 strains of Penicillium roqueforti Thom obtained from culture collections, blue cheeses, sausages, and other sources are shown to grow abundantly on a Czapek Dox liquid medium supplemented with 0.5% acetic acid. None of 30 other strains (including P. charlesii, P. waksmani, P. rugulosum, P. brevi-compactum, P. herquei, P. viridicatum, P. cyclopium, P. velutinum, P. oxalicum, P. toxicarium, P. notatum, P. stoloniferum, P. chrysogenum, P. japonicum, P. casei, P. citreo-viride) exhibited this property. It is suggested that growth on acetic acid provides a simple tool for a rapid and preliminary identification of P. roqueforti Thom since growth can be observed as early as 3 days after inoculation.     

Purple-Pigmented Violacein-Producing <Emphasis Type="Italic">Duganella</Emphasis> spp. Inhabit the Rhizosphere of Wild and Cultivated Olives in Southern Spain

Bacteria have evolved mechanisms that allow them to grow and survive in highly competitive environments like soil and the rhizosphere. Using classical microbiological, physiological, and genetic analyses, we isolated and identified for the first time Duganella spp. associated with the rhizosphere of woody plants in Mediterranean environments that are able to produce violacein, a blue–purple secondary metabolite of considerable biotechnological interest. Based on physiological and biochemical characterization and phylogenetic analysis of different genes including 16S rRNA, gyrB, and vioA (implicated in the synthesis of violacein), the seven Duganella spp. strains isolated and studied were differentiated according to their host of origin (wild versus cultivated olives) and potentially might belong to new species. All the Duganella spp. strains produced violacein in vitro, with natural production levels significantly higher than that previously reported for other violacein-producing bacteria without optimizing growing conditions. The important biological, medical, and industrial applications of violacein make these bacteria good candidates for their biotechnological exploitation because low violacein yields are considered as one of the main limitations of using wild-type strains for extensive exploitation and pigment production. Independent of violacein production, purple-pigmented strains from olives showed proteolytic and lipolytic activities and a weak siderophore production. No in vitro inhibitory activity was demonstrated for bacteria or crude violacein filtrates against plant-pathogenic Gram-negative bacteria and fungi, but they did inhibit Gram-positive bacteria.     

Heat-stability of peroxidase in mycelia of some toxigenic and nontoxigenic Aspergilli and Penicillia

Seven-day-old mycelia from 19 cultures of Aspergillus and 12 cultures of Penicillium were heated to 50, 60, 65, 70, 75, 80, 85, 90 or 95 C for no more than min, and tested for residual peroxidase. The peroxidase from all aspergilli survived heating at 50 through 80 C. Peroxidase from toxigenic strains of Aspergillus flavus, Aspergillus parasiticus and Aspergillus ochraceus survived heating at 85 C and often at 90 C, whereas peroxidase from nontoxigenic strains of A. flavus was inactivated at 90 C and markedly reduced in activity at 85 C. Peroxidase from all penicillia survived heating at all temperatures through 80 C, although the activity of several cultures was reduced at 80 C. Peroxidase activity in mycelia of two strains of Penicillium cyclopium and one of Penicillium puberulum failed to survive heating at 85 C. One strain each of Penicillium roqueforti and Penicillium viridicatum exhibited some peroxidase activity after heating at 90 C, whereas the peroxidase of all other penicillia was inactivated at this temperature.     

Biosynthesis of flavors by Penicillium roqueforti

The development of the unique flavor of blue type cheese depends on the concerted action of numerous enzymes of Penicillium roqueforti involved in protein and lipid metabolism. Protease(s) by degrading casein modify the texture and background flavor of the ripening cheese. Lipase by hydrolyzing milk triglycerides provides flavorful fatty acids and precursors of methyl ketones. The enzyme complex involved in the partial oxidation of free fatty acids and the properties of β-ketoacyl decarboxylase which generates the major flavor components of blue cheese are discussed. Fermentation of P. roqueforti for the rapid production of methyl ketones is briefly reviewed.     

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity – comparable to that obtained with the common preservative calcium propionate (0.3% w/v) – of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.     

Insights into Penicillium roqueforti Morphological and Genetic Diversity

Fungi exhibit substantial morphological and genetic diversity, often associated with cryptic species differing in ecological niches. Penicillium roqueforti is used as a starter culture for blue-veined cheeses, being responsible for their flavor and color, but is also a common spoilage organism in various foods. Different types of blue-veined cheeses are manufactured and consumed worldwide, displaying specific organoleptic properties. These features may be due to the different manufacturing methods and/or to the specific P. roqueforti strains used. Substantial morphological diversity exists within P. roqueforti and, although not taxonomically valid, several technological names have been used for strains on different cheeses (e.g., P. gorgonzolae, P. stilton). A worldwide P. roqueforti collection from 120 individual blue-veined cheeses and 21 other substrates was analyzed here to determine (i) whether P. roqueforti is a complex of cryptic species, by applying the Genealogical Concordance Phylogenetic Species Recognition criterion (GC-PSR), (ii) whether the population structure assessed using microsatellite markers correspond to blue cheese types, and (iii) whether the genetic clusters display different morphologies. GC-PSR multi-locus sequence analyses showed no evidence of cryptic species. The population structure analysis using microsatellites revealed the existence of highly differentiated populations, corresponding to blue cheese types and with contrasted morphologies. This suggests that the population structure has been shaped by different cheese-making processes or that different populations were recruited for different cheese types. Cheese-making fungi thus constitute good models for studying fungal diversification under recent selection.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

Sequence analysis and gene amplification study of the penicillin biosynthesis gene cluster from different strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Industrial strains of Penicillium chrysogenum possess many genomic changes leading to higher levels of penicillin. In this work several production and wild-type strains of Penicillium chrysogenum were used in comparative nucleotide sequence analysis of the biosynthesis cluster. The alignments confirmed sequence conservation not only in promoter regions of the biosynthesis genes but also throughout the entire 44.7-kbp genomic fragment comprising the whole biosynthesis cluster with 15.5-kbp and 13.1-kbp flanking regions. As another titre-enhancing mechanism we subsequently examined gene dosage in two production strains introduced here, NMU2/40 and B14. Quantitative real-time PCR and Southern blot analysis showed the amplification of the biosynthesis genes in both these strains. Through the real-time PCR method the exact copy number was estimated for each of the pcbAB, pcbC and penDE genes. The equal pool of all three genes per genome was confirmed for the both production strains indicating that in these strains the entire penicillin cluster has been amplified as an intact element. Penicillium chrysogenum NMU2/40 was found to carry four copies of the cluster, while six copies were estimated for B14. This also proves the contribution of the additional titre-enhancing mechanisms in both strains, since the industrial data referred much higher production of these strains compared with the single copy reference strain NRRL 1951.     

Controls of the expression of aspA , the aspartyl protease gene from Penicillium roqueforti

The gene (aspA) encoding the extracellular aspartyl protease from Penicillium roqueforti was cloned and characterized. Northern hybridization analyses and β-casein degradation assays revealed that aspA was strongly induced by casein in the medium and efficiently repressed by ammonia. External alkaline pH overrides casein induction, resulting in aspA repression. Cis-acting motifs known to mediate nitrogen and pH regulation of fungal gene expression are present in the aspA promoter and protein-DNA binding experiments showed that mycelial proteins interact with various regions of the promoter. Due to the efficient environmental controls on aspA expression, the promoter of aspA is an attractive candidate for the development of a controllable gene expression system in P. roqueforti. Received: 20 March 1997 / Accepted: 21 June 1997