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.     

Penicillium expansum lipase-catalyzed production of biodiesel in ionic liquids

Penicillium expansum lipase (PEL) was used to catalyze biodiesel production from corn oil in [BMIm][PF₆]¹ (an ionic liquid, IL) and tert-butanol. Both systems were optimized in terms of MeOH/oil molar ratio, reaction temperature, enzyme loading, solvent volume, and water content. The high conversion obtained in the IL (86%) as compared to that in tert-butanol (52%) demonstrates that the IL is a superior solvent for PEL-catalyzed biodiesel production. Poor yields were obtained in a series of hydrophilic ILs. Addition of salt hydrates affected biodiesel production predominantly through the specific ion (Hofmeister) effect. The impact of methanol on both activity and stability of PEL in the IL and in hexane was investigated, in comparison to the results obtained by two commonly used lipases, Novozym 435 and Lipozyme TLIM. The results substantiate that while different lipases show different resistance to methanol in different reaction systems, PEL is tolerant to methanol in both systems.     

Conidiogenones H and I, two new diterpenes of Cyclopiane class from a marine-derived endophytic fungus Penicillium chrysogenum QEN-24S

Two new tetracyclic diterpenes of the rarely reported cyclopiane class, conidiogenones H and I ( 1 and 2 , resp.), along with five related congeners, conidiogenones B – D and F ( 3 – 5 and 6 , resp.) and conidiogenol ( 7 ), were characterized from the culture extracts of Penicillium chrysogenum QEN‐24S, an endophytic fungus derived from an unidentified marine red algal species of the genus Laurencia. The structures of these compounds were established on the basis of extensive spectroscopic analysis. The inhibitory activity of theses diterpenes against four bacteria and one pathogen fungus was evaluated. Conidiogenone B ( 3 ) showed potent activity against Methicillin resistant Staphylococcus aureus (MRSA), Pseudomonas fluorescens, P. aeruginosa, and Staphylococcus epidermidis (each with a MIC value of 8 μg/ml), while conidiogenol ( 7 ) showed obvious activity against P. fluorescens and S. epidermidis (each with a MIC value of 16 μg/ml). This is the first report on antimicrobial activity of cyclopiane diterpenes.     

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.     

<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.     

Penicillium persicinum, a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai province, China

A unique Penicillium isolate from Chinese soil with terverticillate penicilli and ellipsoidal to cylindrical smooth-walled conidia, produces, in addition to the common metabolite ergosterol, copious amounts of an unknown peach-red pigment and the following secondary metabolites: griseofulvin, dechlorogriseofulvin, lichexanthone, roquefortine C, roquefortine D, chrysogine, 2-pyrovoylaminobenzamide, 2-acetyl-quinazolin-4(3H)-one. This isolate, CBS 111235, is described as Penicillium persicinum sp. nov., which belongs to subgenus Penicillium section Chrysogena but is morphologically similar to P. italicum. On the basis of the production of secondary metabolites it resembles P. griseofulvum and P. coprophilum. Sequence data using part of the beta-tubulin gene showed that it is phylogenetically related to P. chrysogenum and P. aethiopicum in section Chrysogena with which it shares both secondary metabolites and ability to grow at 37 degrees C.     

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.     

A novel approach for screening immunogenic proteins in Penicillium marneffei using the DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus

Using serum from guinea-pigs immunized with a DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus to screen a cDNA library of A. fumigatus, we cloned a novel immunogenic 57-kDa protein in A. fumigatus. We also cloned its 55-kDa homologue in Penicillium marneffei, which was possibly related to amino acid biosynthesis and metabolism, with homologues present only in the subphylum Pezizomycotina of Ascomycota. The recombinant 55-kDa protein of P. marneffei reacted strongly with guinea-pig serum immunized with P. marneffei and with the sera of patients with P. marneffei infection. A similar approach could be applied to immunogenic protein screening in other microorganisms for serological diagnosis, epidemiological studies and the study of vaccines.     

The influence of culture conditions on the biosynthesis of secondary metabolites by Penicillium verrucosum Dierck

A Brazilian strain of Penicillium verrucosum was cultivated under different conditions in a two-step process, in order to verify the influence of nutrients, and of time periods of pre-fermentative and fermentative steps on the biosynthesis of metabolites. Extracellular and intracellular extracts were obtained from each culture in the four different production media used. Chemical profiles of the extracts were obtained by HPLC. Extract trypanocidal activities against trypomastigote forms of Trypanosoma cruzi were evaluated. The time period of incubation in the pre-fermentative and fermentative media, as well as the different nutrients tested, qualitatively and quantitatively modified the production of secondary metabolites by P. verrucosum, and the extract trypanocidal activities.