N-linked glycosylation affects catalytic parameters and fluctuation of the active center of Aspergillus awamori exo-inulinase.

Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N-glycosylation, with two of them located near the active center. If expressed in P. verruculosum, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in kcat. These substrates with a short chain of 2-4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N-linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the -1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation.

A Novel Lactam and Two Rare Mycophenolic Acid Derivatives from the Fungus Penicillium sclerotiorum JBHL321.

A novel lactam penicillactam (1), two rare mycophenolic acid (MPA) derivatives penimycophens A and B (2 and 3), together with two known biogenetically related MPA derivatives (4 and 5) and a known alkaloid (6) were isolated from the Penicillium sclerotiorum JBHL321. The structures of these compounds were determined by comprehensive spectroscopic analyses. The absolute configuration of 1 was confirmed by electronic circular dichroism (ECD) calculations. Compound 1 represent the rare example of a oxygen bridge-linked lactam from natural products. Structurally, compounds 2 and 3 were rare MPA derivatives featuring a methoxy group at C-3. The inhibitory activity of compounds 1-6 against two phytopathogenic fungi, three phytopathogenic bacteria and four cancer cell lines were evaluated. Compounds 2-5 exhibited significant cytotoxic activity against HeLa, MCF-7, A549 and MGC-803 cells with IC50 values in the low micromolar to nanomolar. Compound 3 exhibited especially cytotoxic activity against four different cell lines with IC50 values ranging from 0.06 to 0.14 µM, compared to IC50 values ranging from 0.62 to 2.51 µM for epirubicin.

Bioactivity of a dihydroxy flavone produced by Penicillium EU0013 against the wilt pathogen of tomato.

Bioassay-guided isolation of a dihydroxy flavone from the cell extract of Penicillium EU0013 was carried out using preparative LCMS and the structure was interpreted using 1D and 2D NMR spectra. The fungus was fermented in three different nutrient media i.e. Glucose Nutrient Broth, Glucose Peptone Yeast Broth, and Yeast Extract Broth and the cell extract was obtained using organics. Comparative LCMS profiles of the fungus confirmed the enhanced production of dihydroxy flavone in GPYB media. In this study, the pathogenicity of the dihydroxy flavone is reported for the first time against Fusarium oxysporum f.sp. lycopersici, wilt causing pathogen of a tomato plant. In MIC experiments, the compound inhibited the pathogen at 80 µg mL-1. Molecular docking studies of the compound with Fol tomatinase showed five binding interactions. These docking studies may help explore dihydroxy flavones as a lead for developing new fungicides to prevent wilt disease in tomatoes.

DMOA-derived polycyclic meroterpenoids with antiviral activities from the deep-sea-derived fungus Penicillium pancosmium A6A.

Two undescribed 3,5-dimethylorsellinic acid (DMOA) derived meroterpenoids, namely pancosterpenoids A (1) and B (2), were discovered from the EtOAc extract of the deep-sea sediment-derived fungus Penicillium pancosmium A6A. The gross structures were established by detailed analysis of the spectroscopic data (NMR and HRESIMS spectra), while their absolute configurations were resolved by comparing the experimental and calculated ECD data as well as X-ray single crystal diffraction analysis. Pancosterpenoid A (1) was the first representative of DMOA-derived meroterpenoids possessing a 6/6/6/5/5 pentacyclic system, while pancosterpenoid B (2) belongs to a class of rare 13-nor-citreohybridone meroterpenoids. Two metabolites were evaluated the antiviral effects against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) trVLP pseudovirus. As a result, compounds 1 and 2 showed moderately inhibitory activities with IC50 values of 22.37 and 18.12 µM, respectively.

Antimicrobial Isochroman-Derived Atropo-diastereomeric Dimers from Penicillium steckii SCISO 41228.

A pair of unidentified atropisomeric dimers, penicisteckins G (1) and H (2), and twelve known compounds (3-16) were isolated from the marine coral-derived fungus Penicillium steckii SCISO41228. Their structures including the absolute configuration were determined by HR-ESI-MS, ECD, 1D-, and 2D-NMR spectra. Compounds 1 and 2 exhibited potent antibacterial activity against most pathogenic strains, especially for MASA and Micrococcus luteus, with MIC values of 4.0 µg·mL-1. In addition, compounds 2 and 3 exhibit potent antioxidant activity with IC50 values of 10.76 and 8.66 µg·mL-1, respectively.

Safety evaluation of an extension of use of the food enzyme ß-glucosidase from the non-genetically modified Penicillium guanacastense strain AE-GLY.

The food enzyme ß-glucosidase (ß-d-glucoside glucohydrolase, EC 3.2.1.21) is produced with the non-genetically modified Penicillium guanacastense strain AE-GLY by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in four food manufacturing processes. Subsequently, the applicant has requested to extend its use to include three additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. The dietary exposure was calculated to be up to 0.206 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Using the no observed adverse effect level reported in the previous opinion (943 mg TOS/kg bw per day), the Panel derived a margin of exposure of at least 4578. Based on the previous evaluation, the assessment of the new data and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast-famine cycles in a scale-down system.

While traveling through different zones in large-scale bioreactors, microbes are most likely subjected to fluctuating dissolved oxygen (DO) conditions at the timescales of global circulation time. In this study, to mimic industrial-scale spatial DO gradients, we present a scale-down setup based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose-limited chemostat cultures of Penicillium chrysogenum. Such DO feast/famine regime induced a stable and repetitive pattern with a reproducible metabolic response in time, and the dynamic response of intracellular metabolites featured specific differences in terms of both coverage and magnitude in comparison to other dynamic conditions, for example, substrate feast/famine cycles. Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites along with a dynamic and higher redox state (NADH/NAD+) of the cytosol. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased ATP supply. Moreover, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho-hydroxyphenyl acetic acid and degeneration of penicillin productivity. Furthermore, dynamic flux profiles showed the most pronounced variations in central carbon metabolism, amino acid (AA) metabolism, energy metabolism and fatty acid metabolism upon the DO oscillation. Taken together, the metabolic responses of P. chrysogenum to DO gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale-up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

Safety evaluation of the food enzyme endonuclease from the non-genetically modified Penicillium citrinum strain NP 11-15.

The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non-genetically modified Penicillium citrinum strain NP 11-15 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.006 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1010 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 168,333. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Three New Antibacterial Mycophenolic Acid Derivatives from the Marine-Derived Fungus Penicillium sp. HN-66.

Three new mycophenolic acid derivatives, penicacids L-N (1-3), together with four known analogues, were isolated from a fungus Penicillium sp. HN-66 derived from a South China Sea marine sediment. The structures of compounds 1-3 were determined on the basis of HR-ESI-MS, NMR (1H, 13C, HSQC and HMBC) data analyses, and comparison of optical rotations. Antimicrobial activities of 1-7 were tested. The results showed that compounds 1-3 and 5-7 had weak inhibitory effects against E. coli ATCC 25922 with the MIC values of 50 µg/mL.

Penicipyrrolizidinones A-C, three pyrrolizidinone alkaloids with unprecedented skeletons from the mangrove-derived fungus Penicillium sp. DM27.

Three previously undescribed pyrrolizidinone alkaloids, penicipyrrolizidinones A and B (1 and 2), possessing an unprecedented 2-methyl-2-(oct-6-enoyl)pyrrolizidin-3-one skeleton, and penicipyrrolizidinone C (3), featuring a rare 1-alkenyl-2-methyl-pyrrolizidin-3,7-dione skeleton, together with four known pyrrolidine derivatives (4-7) were isolated from the mangrove-derived fungus Penicillium sp. DM27. Their structures were elucidated through comprehensive spectroscopic analysis, theoretical calculations of ECD spectra, and the modified Mosher's method. A plausible biosynthetic pathway for penicipyrrolizidinones A-C (1-3) was proposed. Compounds 4 and 5 exhibited moderate cytotoxicity against B16-F10 melanoma cells with IC50 values of 10.5 µM and 15.5 µM, respectively.

One New Quinazoline-Containing Diketopiperazine Isolated from the Marine-Sponge-Derived Fungus Penicillium sp. SCSIO41043.

One new quinazoline-containing diketopiperazine (1), along with 24 known compounds including nine alkaloids (2-9, and 25), thirteen lactones (10-22), aspterric acid (23), and catechol (24), were isolated from the marine sponge-derived Penicillium sp. SCSIO41043. Their planar structures were unequivocally elucidated by spectroscopic analysis. The absolute configuration of 1 was determined by a comparison of reported and experimental electronic circular dichroism (ECD) spectra. Compound 16 was found to notably inhibit the growth of five pathogenic bacteria and fungi with MIC values ranging from 0.5-16.0 µg/mL. Compounds 7, 17, 20, and 22 demonstrated moderate activity against Micrococcus luteus with MIC values ranging from 35.6 to 71.1 µg/mL. Moreover, 1-3 displayed different degrees of antioxidant activity with EC50 values of 0.98, 0.60, 0.46 mg/mL, respectively.

New Sulfurated Butyrolactones from the Fungus Penicillium janthinellum.

Deep-sea derived fungi are considered as significant resources to discovery structurally diverse and biologically active natural compounds. In this study, four new sulfurated butyrolactones, penijanthiones A-D (1-4), together with four known analogues (5-8), were isolated from a Mariana Trench-derived fungus Penicilliumjanthinellum SH0301. Compounds 1-4 were the undescribed examples for natural butyrolactones coupling with a mercaptolactate moiety. Their structures including the absolute configurations of these new compounds were elucidated by comprehensive spectroscopic data, and calculated electronic circular dichroism (ECD). The plausible biosynthetic pathway of sulfur-incorporation of 1-4 was proposed. All of these isolated compounds were evaluated their cytotoxic, antimicrobial and antiviral activities.

Myco-nanotechnological approach to synthesize gold nanoparticles using a fungal endophyte, Penicillium oxalicum and unravelling their antibacterial activity and anti-breast cancer role via metabolic reprogramming.

The present study has been designed to fabricate fungal endophyte assisted gold nanoparticles and elucidate their anti-breast cancer potential. The aqueous extract of fungal endophyte, Penicillium oxalicum, associated with the medicinal plant Amoora rohituka has been used for the fabrication of gold nanoparticles (POAuNPs). The physicochemical characterization using UV-Vis spectroscopy, FTIR, XRD, DLS, Zeta potential, TEM and FESEM analysis revealed stable, uniform distribution, spherical-shape and crystalline nature of POAuNPs with size range of 3-46 nm. Further, POAuNPs potentially inhibited the growth of pathogenic bacterial strains, E. coli and S. aureus. The synthesized POAuNPs has shown potential antioxidant effects against DPPH, superoxide and nitric oxide radical scavenging assay with an EC50 value of 8.875±0.082, 52.593±2.506 and 43.717±1.449 µg/mL, respectively. Moreover, the value of EC50 for total antioxidant capacity of POAuNPs was found to be 23.667±1.361 µg/mL. The cell viability of human breast cancer cells, MDA-MB-231 and MCF-7 was found to be reduced after treatment with POAuNPs and IC50 values were found to be 19.753±0.640 and 35.035±0.439 µg/mL respectively. Further, in vitro biochemical assays revealed POAuNPs induced metabolic reprogramming in terms of reduced glucose uptake and increased LDH release and, disruption of oxidative balance through depletion of GSH level, increased nitric oxide level and lipid peroxidation as a possible pathway to suppress the human breast cancer cell proliferation. Apoptosis-specific nuclear modulations induced by POAuNPs in human breast cancer cells were validated through DAPI nuclear staining. The present investigation thus attempted to show first ever fabrication of gold nanoparticles using aqueous extract of P. oxalicum associated with A. rohituka. The results revealed unique physico-chemical characteristics of myogenic gold nanoparticles and screening their effect against breast cancer via metabolic reprogramming and induction of apoptosis thus adds great significance against cancer therapeutics, suggesting further exploration to develop nanotherapeutic drugs.

Steroids and Epicoccarines from Penicillium aurantiancobrunneum.

Lichens are symbiotic organisms comprised of mycobionts and photobiont partners. They are known to produce bioactive secondary metabolites and most of these are biosynthesized by mycobionts. Investigations of cultures of isolated lichen-associated fungi have shown promise for the discovery of cytotoxic compounds. Thus, the lichen-associated fungus Penicillium aurantiacobrunneum was studied for its potential to produce novel compounds and the new sterols (20S*)-hydroxy-24(28)-dehydrocampesterol (1), 7α-methoxy-8ß-hydroxypaxisterol (2), 14-nor-epicoccarine A (3) and 14-nor-epicoccarine B (4), as well as the known compound PF1140 (5), were isolated. The structures of these compounds were elucidated using methods including nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). Following cytotoxicity assays, compound 1 demonstrated activity against the pancreatic adenocarcinoma epithelial HPAC cell line at 17.76 ± 5.35 µM. Since the structures of compounds 3 and 4 were very similar to tetramic acid derivatives that were reported to be biosynthesized from a polyketide synthase- non-ribosomal peptide synthetase (PKS-NRPS) hybrid pathway, a plausible biosynthetic route for production in P. aurantiacobrunneum is proposed herein.

Cercospora leaf spot impacts on postharvest disease and respiration of affected sugarbeet roots.

In Michigan, sugarbeets (Beta vulgaris) are stored for up to 200 days post-harvest, during which sugar loss may occur due to energy use from respiration and factors like rot. Cercospora leaf spot (CLS) has been considered a potential predisposing factor for increased storage rot. To investigate these impacts, field and postharvest studies evaluated storage rot symptom development in sugarbeets with designated 'high' or 'low' in-season CLS severity. Root slices of sugarbeets from each CLS level were inoculated with Fusarium graminearum, Botrytis cinerea, or Penicillium vulpinum and symptoms assessed after seven days. Across three CLS-susceptible commercial varieties, there were no significant differences between storage rot susceptibility to any of the tested pathogens in hand-harvested sugarbeets, regardless of CLS level, at any storage timepoint in 2020 or 2021 (P > 0.05). In studies using CLS-susceptible and -resistant germplasm and varieties, CLS effects were inconsistent and only significant in one parameter at two of six storage timepoints across years (P < 0.05). Across storage pathogens, prior CLS level also did not impact root respiration or the change in respiration rate from initial to final storage timepoint in either 2021 or 2023 (P > 0.05). Of note, B. cinerea caused more severe symptoms than other pathogens in these studies (P < 0.05). Finally, varietal responses differed significantly to storage pathogens (P < 0.05) and may be of interest to future cultivar development efforts. This research increases our understanding of factors contributing to potential storage losses, which will improve yield and profit for sugar growers.

A new tetrahydroxybenzophenone from a marine-associated fungus of penicillium sp. and its potential antibacterial activity.

Penicillium species are renowned for their ability to produce a wide range of secondary metabolites with medicinal properties. In this study, compounds 1-10 were isolated from Penicillium sp. Z-16, of which compound 1 is a new benzophenone derivative named methyl 2-(2,6-dihydroxy-4-methylbenzoyl)-4,5-dihydroxy-3-methoxybenzoate. The chemical structure of 1 was determined through comprehensive spectroscopic analysis, including 1D, 2D NMR (HMBC, HSQC) and HRESIMS. In addition, six other known compounds (11-16) were isolated and identified from Penicillium sp. T-5-1. The antimicrobial activity tests demonstrated that compound 1 was moderately active against Candida albicans with a MIC value of 125 µg/mL, while compound 2 showed a MIC value of 62.5 µg/mL against Staphylococcus aureus.

Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea.

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.

Combination of Cinnamaldehyde/ß-cyclodextrin inclusion complex and L-phenylalanine effectively reduces the postharvest green mold in citrus fruit.

The essential oil and ß-cyclodextrin inclusion complex was able to inhibit the growth of Penicillium digitatum, a damaging pathogen that causes green mold in citrus fruit. In this study, cinnamaldehyde-ß-cyclodextrin inclusion complex (ß-CDCA) for controlling citrus green mold was synthesized by the co-precipitation method. Characterization of ß-CDCA revealed that the aromatic ring skeleton of cinnamaldehyde (CA) was successfully embedded into the cavity of ß-CD to form the inclusion complex. ß-CDCA inhibited P. digitatum at a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 4.0 g/L. FT-IR spectroscopy analysis, calcofluor white staining, extracellular alkaline phosphatase (AKP) activity and propidium iodide (PI) staining of hyphae morphology showed that ß-CDCA may damage the cell ultrastructure and membrane permeability of P. digitatum. The study further demonstrated that hydrogen peroxide (H2O2), malondialdehyde (MDA), and reactive oxygen species (ROS) markedly accumulated in 1/2 MIC ß-CDCA treated hyphae. This implied that ß-CDCA inhibited growth of P. digitatum by the triggering oxidative stress, which may have caused cell death by altering cell membrane permeability. In addition, in vivo results showed that ß-CDCA alone or combined with L-phenylalanine (L-PHe) displayed a comparable level to that of prochloraz. Therefore, ß-CDCA combined with L-PHe can thus be used as an eco-friendly preservative for the control green mold in postharvest citrus fruit.

Antibacterial Indole-diterpenoid Alkaloids from the Marine Fungus Penicillium sp. ZYX-Z-718.

Two new indole-diterpenoids, penpaxilloids F and G (1 and 2), along with 11 known analogues (3-13), were isolated from the marine fungus Penicillium sp. ZYX-Z-718. The structures of the new compounds were identified by extensive spectroscopic analyses including HR-ESI-MS, UV, and NMR, as well as theoretical NMR chemical shifts and ECD calculations. Compounds 6 and 10 showed antibacterial activity against Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis, and MRSA with MIC values ranging from 16.0 to 32.0 µg/mL.

New Phthalides and Isochromanone Derivatives from the Deep-Sea-Derived Fungus Penicillium bialowiezense A3.

Two new phthalide derivatives, namely bialowalides A (1) and B (8), and one new isochromanone analogue biourgalide C (11), together with 8 known phthalides (2‒7, 9, and 10) as well as two known isochromanones (12 and 13) were discovered from the EtOAc extract of the deep-sea-derived fungus Penicillium bialowiezense A3. The structures were resolved on the basis of extensive spectroscopic analyses (NMR and HRESIMS data), in association with the modified Mosher's method and ECD data for the determination of the absolute configurations. All isolated secondary metabolites (1‒13) were tested for their antiviral activities against the SARS-CoV-2 trVLP pseudovirus at a concentration of 25 µM. As a result, compounds 1, 5, 11, and 12 exhibited the inhibitory effects against the luminescence at 46.2%, 39.6%, 45.5%, and 48.8%, respectively.