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.     

Resolution of four large chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome 1 (10.4 Mb) in Penicillium chrysogenum

Four chromosomes were resolved by pulsed field gel electrophoresis in Penicillium notatum (10.8, 9.6, 6.3 and 5.4 Mb in size) and in five different strains of Penicillium chrysogenum (10.4, 9.6, 7.3 and 6.8 Mb in the wild type). Small differences in size were found between the four chromosomes of the five P. chrysogenum strains. The penicillin gene cluster was localized by hybridization with a pcbAB probe to chromosome II of P. notatum and to chromosome I of all P. chrysogenum strains except the deletion mutant P. chrysogenum npe10, which lacks this DNA region. The pyrG gene was localized to chromosome I in P. notatum and to chromosome II in all P. chrysogenum strains except in the mutant AS-P-78 where the probe hybridized to chromosome 111. A major chromosomal rearrangement seems to have occurred in this high penicillin producing strain. A fast moving DNA band observed in all gels corresponds to mitochondrial DNA. The total genome size has been calculated as 32.1 Mb in P. notatum and 34.1 Mb for the P. chrysogenum strains.     

Aroma production by spores ofPenicillium roqueforti on a synthetic medium

Summary The synthesis of 2-heptanone from the sodium salt of octanoic acid by spores of five strains ofPenicillium roqueforti was studied. The strains showed a high disparity in kinetic behavior. The one selected, which was originally isolated from blue cheese, had a good resistance to substrate inhibition along with a good apparent biotransformation yield (close to 60%). An activator was needed in the incubation medium. The loss of activity of aging spores was reduced by the activator compounds; ethanol exhibited the highest efficiency. When spores were produced on buckwheat seeds with a solid state fermentation technique, the medium itself was an activator source. When the biotransformation reaction was carried out in a stirred aerated fermentor, the volatile loss by air-stream stripping had to be taken into account. No ketone metabolism occurred with the strain used.     

Mycotoxins from the fungi Penicillium vulpinum (Cooke & Massee) Seifert & Samson

Mycotoxins produced by seven strains of Penicillium vulpinum (formerly Penicillium claviforme) isolated from different sources were studied. The strains were characterized by specific profiles of secondary metabolites and produced mycotoxins of different structural types. In addition to toxins already known for this fungal species (patulin, roquefortine, 3,12-dihydroroquefortine, oxalin, viridicatin, cyclopenin, and alpha-cyclopiazonic acid), the strains studied also produced indolyl-3-acetic acid, griseofulvin, meleagrin, and cyclopeptin.     

The Penicillium commune Thom and Penicillium clavigerum Demelius Fungi Producing Fumigaclavines A and B

The type strains Penicillium clavigerum VKM F-447 and P. commune VKM F-3233 are found to produce fumigaclavines A and B. Of the seven other strains of these species, only two strains, P. commune VKM F-3088 and F-3491, possess the ability to synthesize these alkaloids. It is suggested that the five other strains under study either lost such an ability or require very specific conditions for the synthesis of these alkaloids.     

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

Summary Penicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.     

<Emphasis Type="Italic">Penicillium frequentans</Emphasis> isolated from<Emphasis Type="Italic"> Picea glehnii</Emphasis> seedling roots as a possible biological control agent against damping-off

Picea glehnii seedlings are affected by damping-off fungi in nurseries. The aims of this study were (1) to isolate fungi grown in the seedling rhizosphere in forest soil of P. glehnii, (2) to select fungi that produce antifungal compounds against Pythium vexans, and (3) to examine whether or not selected fungi can protect seedlings from P. vexans. Penicillium frequentans from Picea glehnii seedling roots produced antibiotic penicillic acid. Penicillic acid did not cause significant phytotoxicity to the seedlings. Penicillium frequentans increased the average percentage of surviving seedlings when inoculated together with Pythium vexans, but the increase was not significant. Vigorous mycelial growth of P. frequentans around seedling roots seems to be one of the mechanisms for protection, but the amount of penicillic acid was too low to show antifungal activity in the seedling rhizosphere.     

Growth Characteristics and Glucose Oxidase Production in Mutant Penicillium funiculosum Strains

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th hour of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.     

Random amplified polymorphic DNA (RAPD) analysis ofPenicillium marneffei strains isolated from AIDS patients in Thailand

Results of random amplified polymorphic DNA (RAPD) analysis using three different primers showed that 16 strains ofPenicillium marneffei isolated from AIDS patients in Thailand belonged to a genetically homogenous group, but different slightly from an isolate from bamboo rat in China. Six PCR fragments (from about, 200 to 600 bp) that were commonly observed in the RAPD fingerprint of all strains were extracted and sequented. Usefulness of this sequence information for identification ofP. marneffei is discussed.     

Laccase activities of a soil fungus Penicillium simplicissimum in relation to lignin degradation

Summary The laccase activities of Penicillium simplicissimum H5 during solid-state fermentation with rice straw were studied. Degradation of lignocellulose was also followed. Results showed that all supplemental carbon sources inhibited the laccase activity in different degrees, while suitable concentrations of supplemental nitrogen sources remarkably enhanced the laccase activity. The enhancement of activity by the ordinary laccase inducers 2, 2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and xylidine was not observed in this study. Lignocellulose degradation was improved when laccase activity was relatively low, suggesting a polymerizing function of laccase in lignin degradation by P. simplicissimum.     

Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

The Biosynthesis of Low-Molecular-Weight Nitrogen-Containing Secondary Metabolites—Alkaloids—by the Resident Strains of Penicillium chrysogenum and Penicillium expansum Isolated on Board the Mir Space Station

The analysis of the absorption spectra of the low-molecular-weight nitrogen-containing secondary metabolites—alkaloids—of four Penicillium chrysogenum strains and six P. 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 acetyltryptamine, and a yellow metabolite containing the benzoquinone chromophore).     

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     

Production of lipase free of citrinin by Penicillium citrinum

Lipase (Glycerol ester hydrolase E.G. 3.1.1.3) from a Brazilian strain of Penicillium citrinum free of the mycotoxin citrinin has been investigated. Citrinin production was inhibited by using culture medium containing olive oil, soybean oil and corn oil as carbon sources. Potassium concentration and pH play an important role in citrinin production. Potassium concentration lower than 30 mM and pH below 4.5 inhibited the mycotoxin production. P. citrinum produced lipase free of extraneous proteins and citrinin when cultured using, as nitrogen source, ammonium sulphate (lipase activity of 7.88 U/mg) and yeast extract (lipase activity of 4.95 U/mg) with olive oil as carbon source. This data is relevant to the larger scale production of lipases for food technology applications, from Penicillium citrinum.     

The isoepoxydon dehydrogenase gene of the patulin metabolic pathway differs for Penicillium griseofulvum and Penicillium expansum

Purified DNA from isolates of Penicillium griseofulvum and P. expansum was used as a template to amplify a 600-bp fragment of the isoepoxydon dehydrogenase (idh) gene of the patulin biosynthetic pathway. Primer pairs designed from the P. griseofulvum gene (GenBank accession AF006680) to amplify specific regions of the idh gene yielded similar-sized bands for all strains. Asymmetrical amplification produced DNA products for sequencing and DNA sequences were translated to produce the corresponding amino acid sequences. After removal of two introns present in the region sequenced, amino acid sequences were compared. There were 12 amino acid differences between P. expansum and P. griseofulvum in the coding region. The differences correlated with the amount of patulin previously produced in culture, with strains of P. griseofulvum producing the greatest amounts of patulin.     

草酸青霉的植物多糖降解酶基因表达调控的研究进展

植物生物质是地球上最丰富的可再生资源,对其生物炼制可生产高附加值的生物基产品。生物炼制需要使用植物多糖降解酶(plant-polysaccharide-degrading enzymes,PPDEs),如纤维素酶、木聚糖酶和生淀粉酶。丝状真菌草酸青霉(Penicillium oxalicum)能分泌完整的具有高活力的植物多糖降解酶,但其产量低限制了大规模生产及应用。草酸青霉中植物多糖降解酶的生物合成受到多种调控因子包括转录因子的严格调控。本文主要介绍在以植物生物质甘蔗渣和木薯生淀粉为原料的生物炼制中,涉及的一些关键微生物方面的问题,如从高产植物多糖降解酶的真菌菌株的筛选、育种,到草酸青霉植物多糖降解酶合成及其基因表达的调控基因的鉴定,以及酶产量提高的工程菌株的构建等,为丝状真菌资源的开发与利用提供理论指导。     

Novel cellulases from an extremophilic filamentous fungi <Emphasis Type="Italic">Penicillium citrinum</Emphasis>: production and characterization

The enzymatic hydrolysis of cellulose has potential economical and environment-friendly applications. Therefore, discovery of new extremophilic cellulases is essential to meet the requirements of industry. Penicillium citrinum (MTCC 6489) that was previously isolated from soil in our laboratory, produced alkali tolerant and thermostable cellulases. Endoglucanase and filter paper activity hydrolase (FPAse) production of P. citrinum were studied using wheat bran substrate in solid state and submerged culture. Zymogram analysis of endoglucanase revealed the presence of two isoforms differing in molecular weight. One of them was 90 kDa and other one was 38 kDa. Partially purified endoglucanase showed two different peaks at pH 5.5 and 8.0, respectively, in its pH optima curve. But FPase showed only one peak (at pH 6.5) in its pH optima curve. Cellulase of P. citrinum is thermostable in nature. The present work reports for the first time, the alkali stable cellulase from alkali tolerant fungus Penicillium citrinum. Thermostable endoglucanase from P. citrinum may have potential effectiveness as additives to laundry detergents.     

Identification of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> in Paraffin-Embedded Tissue Using Nested PCR

Penicillium marneffei is one of the unique thermally dimorphic fungi in Penicillium species that causes a disseminated, progressive and life threatening infection in immunocompromised patients. The diagnosis of Penicilliosis marneffei depends on culture that may delay the treatment due to the time-consuming process. In the present study, we evaluated the specificity and sensitivity of nested PCR to identify Penicillium marneffei from paraffin-embedded tissue. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA of Penicillium marneffei. The outer primers (RRF1 and RRH1) were specific to fungi. The inner primers (Pm1 and Pm2) were specific to Penicillium marneffei. The specific fragment of approximately 400 bp was amplified from all paraffin-embedded tissues from 14 patients with Penicilliosis marneffei and 10 bamboo rats. The detectable DNA concentration of single PCR and nested PCR were 14 pg/μl and 14 fg/μl, respectively. Further studies are required in order to use nested PCR for early diagnosis of the disease.     

The effects of Penicillium digitatum and Fusarium oxysporum rots on nutritional content of pawpaw (Carica papaya L.)

Investigations were carried out on the effects of Penicillium digitatum and Fusarium oxysporum on the nutritional value of pawpaw (Carica papaya). Decreases were observed in ash content, phosphorus, sodium, reducing sugars and ascorbic acid levels of fruits infected with P. digitatum, but increases in calcium and potassium content. In fruits infected by F. oxysporium, there were decreases in phosphorus, calcium, sodium ascorbic acid and reducing sugar levels; but the levels of ash content increased. The total protein level increased in the fruits infected with both fungi. These results revealed a reduction in fruit quality.