Titer improvement of mycophenolic acid in the novel producer strain Penicillium arizonense and expression analysis of its biosynthetic genes.

Mycophenolic acid (MPA) is the active ingredient in the most important immunosuppressive pharmaceuticals. It has antifungal, antibacterial, antiviral, anti-psoriasis, and antitumor activities. Therefore, its overproduction in addition to gene expression analysis was our main target. Through this study, we isolated a novel potent mycophenolic acid (MPA) producer strain of the genus Penicillium from the refrigerated Mozzarella cheese and it was identified with the molecular marker ITS and benA genes as P. arizonenseHEWt1. Three MPA overproducer mutants were isolated by exposing the wild type to different doses of gamma-rays, and the fermentation conditions for the highest production of MPA were optimized. The results indicated that MPA amounts produced by the mutants MT1, MT2, and MT3 were increased by 2.1, 1.7, and 1.6-fold, respectively, compared with the wild-type. The growth of both mutant and wild-type strains on PD broth, adjusted to pH 6 and incubated at 25 °C for 15 d, were the best conditions for maximum production of MPA. In a silico study, five orthologs genes of MPA biosynthesizing gene clusters in P. brevicompactum were predicted from the genome of P. arizonense. Sequencing and bioinformatic analyses proved the presence of five putative genes namely mpaA, mpaC, mpaF, mpaG, and mpaH in the P. arizonense HEWt1 genome. Gene expression analysis by qRT-PCR indicated an increase in the transcription value of all annotated genes in the three mutants over the wild type. A highly significant increase in the gene expression of mpaC, mpaF, and mpaH was observed in P. arizonense-MT1 compared with wild-type. These results confirmed the positive correlation of these genes in MPA biosynthesis and are the first report regarding the production of MPA by P. arizonense.Kew word.Mycophenolic acid, Penicillium arizonense, mutagenesis, gene expression.

Genome-wide identification of the Penicillium digitatum bZIP gene family and the roles of one key member, PdatfA.

Penicillium digitatum is the most common cause of postharvest decay in citrus fruits around the world. Previous studies revealed that the bZIP gene family plays crucial roles in development, stress adaptation, and pathogenicity in fungi. However, little is known about the bZIP genes in P. digitatum. In this study, we systematically identified the bZIP family in 23 Penicillium species and analyzed their evolutionary relationships. We found that gene loss and gene duplication shaped the evolution of the Penicillium bZIP family. P. digitatum experienced 3 bZIP gene loss events, but with no gene duplication. We subsequently characterized the biological functions of one important member, PdatfA in P. digitatum by constructing the deletion mutant. Results showed that ΔPdatfA exhibited a moderate growth defect, reduced pigmentation, and slightly increased resistance to fungicides iprodione and fludioxonil. However, ΔPdatfA displayed similar rot symptoms to that of the wild-type. The ΔPdatfA mycelia were not affected in response to oxidative stress while its conidia showed enhanced resistance due to the upregulation of catalases. Our results provide new insights into the evolution and functions of the bZIP gene family in Penicillium.

Production of Mycophenolic Acid by a Newly Isolated Indigenous Penicillium glabrum.

Soil-occupant fungi produce a variety of mycotoxins as secondary metabolites, one of which is mycophenolic acid (MPA), an antibiotic and immunosuppressive agent. MPA is mainly produced by several species of Penicillium, especially Penicillium brevicompactum. Here, we present the first report of MPA production by a local strain belonging to Penicillium glabrum species. We screened ascomycete cultures isolated from moldy food and fruits, as well as soils, collected from different parts of Iran. MPA production of one hundred and forty Penicillium isolates was analyzed using HPLC. Three MPA producer isolates were identified, among which the most producer was subjected to further characterization, based on morphological and microscopic analysis, as well as molecular approach (ITS, rDNA and beta-tubulin gene sequences). The results revealed that the best MPA producer belongs to P. glabrum IBRC-M 30518, and can produce 1079 mg/L MPA in Czapek-Dox medium.

Anti-Inflammatory Azaphilones from the Edible Alga-Derived Fungus Penicillium sclerotiorum.

To discover the new medical entity from edible marine algae, our continuously natural product investigation focused on endophytes from marine macroalgae Grateloupia sp. Two new azaphilones, 8a-epi-hypocrellone A (1), 8a-epi-eupenicilazaphilone C (2), together with five known azaphilones, hypocrellone A (3), eupenicilazaphilone C (4), ((1E,3E)-3,5-dimethylhepta-1,3-dien-1-yl)-2,4-dihydroxy-3-methylbenzaldehyde (5), sclerotiorin (6), and isochromophilone IV (7) were isolated from the alga-derived fungus Penicillium sclerotiorum. The structures of isolated azaphilones (1-7) were elucidated by spectrometric identification, especially HRESIMS, CD, and NMR data analyses. Concerning bioactivity, cytotoxic, anti-inflammatory, and anti-fibrosis activities of those isolates were evaluated. As a result, compound 1 showed selective toxicity toward neuroblastoma cell line SH-SY5Y among seven cancer and one fibroblast cell lines. 20 µM of compounds 1, 3, and 7 inhibited the TNF-α-induced NFκB phosphorylation but did not change the NFκB activity. Compounds 2 and 6 respectively promoted and inhibited SMAD-mediated transcriptional activities stimulated by TGF-ß.

Bioguided Isolation of Cyclopenin Analogues as Potential SARS-CoV-2 Mpro Inhibitors from Penicillium citrinum TDPEF34.

SARS-CoV-2 virus mutations might increase its virulence, and thus the severity and duration of the ongoing pandemic. Global drug discovery campaigns have successfully developed several vaccines to reduce the number of infections by the virus. However, finding a small molecule pharmaceutical that is effective in inhibiting SARS-CoV-2 remains a challenge. Natural products are the origin of many currently used pharmaceuticals and, for this reason, a library of in-house fungal extracts were screened to assess their potential to inhibit the main viral protease Mpro in vitro. The extract of Penicillium citrinum, TDPEF34, showed potential inhibition and was further analysed to identify potential Mpro inhibitors. Following bio-guided isolation, a series of benzodiazepine alkaloids cyclopenins with good-to-moderate activity against SARS-CoV-2 Mpro were identified. The mode of enzyme inhibition of these compounds was predicted by docking and molecular dynamic simulation. Compounds 1 (isolated as two conformers of S- and R-isomers), 2, and 4 were found to have promising in vitro inhibitory activity towards Mpro, with an IC50 values range of 0.36-0.89 µM comparable to the positive control GC376. The in silico investigation revealed compounds to achieve stable binding with the enzyme active site through multiple H-bonding and hydrophobic interactions. Additionally, the isolated compounds showed very good drug-likeness and ADMET properties. Our findings could be utilized in further in vitro and in vivo investigations to produce anti-SARS-CoV-2 drug candidates. These findings also provide critical structural information that could be used in the future for designing potent Mpro inhibitors.