Phoenicin Switch: Discovering the Trigger for Radical Phoenicin Production in Multiple Wild-Type Penicillium Species.

Society faces the challenge of storing energy from sustainable sources in inexpensive, nontoxic ways that do not deplete the limited resources of Earth. In this regard, quinone redox flow batteries have been proposed as ideal; however, industrially used quinones have traditionally been synthesized from fossil fuels. Therefore, we investigated the production of phoenicin (compound 1), a deep violet dibenzoquinone produced by certain Penicillium species, for its industrial potential. Strains grew as surface cultures on customized growth media with varying production parameters, and phoenicin production was assessed by ultrahigh-performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOF MS) analysis of the supernatant. Phoenicin production was reliant on the sucrose concentration, and by varying that, we produced 4.94 ± 0.56 g/L phoenicin on a Czapek yeast autolysate broth (CY)-based medium with Penicillium phoeniceum (CBS 249.32) as the production host, with 71.91% phoenicin purity in the resulting medium broth. Unexpectedly, metabolites corresponding to phoenicin polymers were tentatively identified in P. phoeniceum, of which the dimer (diphoenicin) was a major chromatographic peak. An MS-based metabolomics study was conducted on P. atrosanguineum using feature-based molecular networking and multivariate statistics, and it was found that few or no known secondary metabolites besides phoenicin were secreted into the growth medium. Finally, the effects of sucrose, sodium nitrate, and yeast extract (YE) in the growth medium were investigated in a 23 full factorial design. The results indicated an optimal sucrose concentration of 92.87 g/L on CY when NaNO3 and YE were fixed at 3 and 5 g/L, respectively. IMPORTANCE This work was undertaken to explore the production of fungal quinones in wild-type strains for use as electrolytes in redox flow batteries. As society converts energy production in a more sustainable direction, it becomes increasingly more important to store sustainable energy in smart ways. Conventional battery technologies imply the use of highly toxic, expensive, and rare metals; thus, quinone redox flow batteries have been proposed to be a desirable alternative. In this study, we explored the possibility of producing the fungal quinone phoenicin in Penicillium spp. by changing the growth parameters. The production of other secondary metabolites and known mycotoxins was also investigated in a metabolomics study. It was shown that phoenicin production was activated by optimizing the carbon concentration of the medium, resulting in high titers and purity of the single metabolite.

Secondary metabolite production by cereal-associated penicillia during cultivation on cereal grains.

Cereals are vulnerable substrates for fungal growth and subsequent mycotoxin contamination. One of the major fungal genera to colonize the ecosystem of stored grain is Penicillium, especially species in the series of Viridicata and Verrucosa. Culturing these species on grains, we hoped to induce the production of relevant secondary metabolites produced by these fungi in the early stage of cereal breakdown. In a multivariate setup six different cereal grains (wheat, rye, barley, oat, rice, and maize), one kind of white beans, and two standard fungal media, Yeast Extract Sucrose agar (YES agar) and Czapek Yeast Autolysate agar (CYA agar), were inoculated with the ten most important cereal-associated species from Penicillium (P. aurantiogriseum, P. cyclopium, P. freii, P. melanoconidium, P. neoechinulatum, P. polonicum, P. tricolor, P. viridicatum, P. hordei, and P. verrucosum). P. nordicum is a meat-associated species, which was included due to its chemical association with P. verrucosum, in addition to see if a substrate change would alter the profile of known chemistry. We found that cereals function very well as substrates for secondary metabolite production, but did not present significantly different secondary metabolite profiles, concerning known chemistry, as compared to standard laboratory agar media. However, white beans altered the semi-quantitative secondary metabolite profiles for several species. Correlations between substrates and certain metabolites were observed, as illuminated by principal component analysis. Many bioactive secondary metabolites were observed for the first time in the analyzed fungal species, including ergot type alkaloids in P. hordei.

The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius.

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74-96 % compared to the wild type. The endoglucanase activity was reduced with 51-78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.

Name changes in medically important fungi and their implications for clinical practice.

Recent changes in the Fungal Code of Nomenclature and developments in molecular phylogeny are about to lead to dramatic changes in the naming of medically important molds and yeasts. In this article, we present a widely supported and simple proposal to prevent unnecessary nomenclatural instability.

Reconstitution of biosynthetic machinery for the synthesis of the highly elaborated indole diterpene penitrem.

Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation-initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two-step P450-catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.

Aggressive dereplication using UHPLC-DAD-QTOF: screening extracts for up to 3000 fungal secondary metabolites.

In natural-product drug discovery, finding new compounds is the main task, and thus fast dereplication of known compounds is essential. This is usually performed by manual liquid chromatography-ultraviolet (LC-UV) or visible light-mass spectroscopy (Vis-MS) interpretation of detected peaks, often assisted by automated identification of previously identified compounds. We used a 15 min high-performance liquid chromatography-diode array detection (UHPLC-DAD)-high-resolution MS method (electrospray ionization (ESI)(+) or ESI(-)), followed by 10-60 s of automated data analysis for up to 3000 relevant elemental compositions. By overlaying automatically generated extracted-ion chromatograms from detected compounds on the base peak chromatogram, all major potentially novel peaks could be visualized. Peaks corresponding to compounds available as reference standards, previously identified compounds, and major contaminants from solvents, media, filters etc. were labeled to differentiate these from compounds only identified by elemental composition. This enabled fast manual evaluation of both known peaks and potential novel-compound peaks, by manual verification of: the adduct pattern, UV-Vis, retention time compared with log D, co-identified biosynthetic related compounds, and elution order. System performance, including adduct patterns, in-source fragmentation, and ion-cooler bias, was investigated on reference standards, and the overall method was used on extracts of Aspergillus carbonarius and Penicillium melanoconidium, revealing new nitrogen-containing biomarkers for both species.

A novel contaminant in museums? A cross-sectional study on xerophilic Aspergillus growth in climate-controlled repositories.

In the last decade, extensive fungal growth has developed in Danish museums parallel to climate change, challenging occupational health and heritage preservation. The growth was unexpected as the museums strived to control relative humidity below 60 %, and it should exceed 75 % to risk growth. A Danish case study found xerophilic Aspergillus species able to grow at low relative humidity in a museum repository. This cross-sectional study aimed to examine whether xerophilic growth from Aspergillus section Restricti has become a novel contaminant nationally distributed in Danish museum repositories striving to control relative humidity according to international environmental recommendations for heritage collections. The study examined The National Museum of Denmark and eight large State Recognized museums distributed throughout Denmark. It was based on 600 swab and tape-lift samples and 60 MAS100-Eco and filter air samples analyzed for fungi with cultivation and morphological identification, Big-Dye-Sanger sequencing, CaM-Nanopore and ITS-Illumina amplicon sequencing. The study showed growth from seven xerophilic Aspergillus species: A. conicus, A. domesticus, A. glabripes, A. halophilicus, A. magnivesiculatus, A. penicilloides, A. vitricola, of which three are new to Denmark, and 13 xerotolerant Aspergillus species. There was no growth from other fungal species. The multiple detection approach provided a broad characterization; however, there was variance in the detected species depending on the analysis approach. Cultivation and Big-Dye Sanger sequencing showed the highest Aspergillus diversity, detecting 17 species; CaM-Nanopore amplicon sequencing detected 12 species; and ITS-illumina amplicon sequencing detected two species but the highest overall diversity. Cultivation, followed by Big-Dye Sanger and CaM-amplicon sequencing, proved the highest compliance. The study concluded that xerophilic Aspergillus growth is nationally distributed and suggests species from Aspergillus section Restricti as a novel contaminant in climate-controlled museum repositories. To safeguard occupational health and heritage preservation research in sustainable solutions, avoiding xerophilic growth in museum collections is most important.

Anticancer and antifungal compounds from Aspergillus, Penicillium and other filamentous fungi.

This review covers important anticancer and antifungal compounds reported from filamentous fungi and in particular from Aspergillus, Penicillium and Talaromyces. The taxonomy of these fungi is not trivial, so a focus of this review has been to report the correct identity of the producing organisms based on substantial previous in-house chemotaxonomic studies.

Bio-activity and dereplication-based discovery of ophiobolins and other fungal secondary metabolites targeting leukemia cells.

The purpose of this study was to identify and characterize fungal natural products (NPs) with in vitro bioactivity towards leukemia cells. We based our screening on a combined analytical and bio-guided approach of LC-DAD-HRMS dereplication, explorative solid-phase extraction (E-SPE), and a co-culture platform of CLL and stromal cells. A total of 289 fungal extracts were screened and we tracked the activity to single compounds in seven of the most active extracts. The novel ophiobolin U was isolated together with the known ophiobolins C, H, K as well as 6-epiophiobolins G, K and N from three fungal strains in the Aspergillus section Usti. Ophiobolins A, B, C and K displayed bioactivity towards leukemia cells with induction of apoptosis at nanomolar concentrations. The remaining ophiobolins were mainly inactive or only slightly active at micromolar concentrations. Dereplication of those ophiobolin derivatives possessing different activity in combination with structural analysis allowed a correlation of the chemical structure and conformation with the extent of bioactivity, identifying the hydroxy group at C3 and an aldehyde at C21, as well as the A/B-cis ring structure, as indispensible for the strong activity of the ophiobolins. The known compounds penicillic acid, viridicatumtoxin, calbistrin A, brefeldin A, emestrin A, and neosolaniol monoacetate were identified from the extracts and also found generally cytotoxic.

Production of Fungal Quinones: Problems and Prospects.

Fungal quinones can be used for a variety of applications, such as pharmaceuticals, food colorants, textile dyes, and battery electrolytes. However, when producing quinones by fungal cultivation, many considerations arise regarding the feasibility of a production system, such as the quinone yield, purity, ease of extraction, and the co-production of mycotoxins. In this work, we display the initial screening of filamentous fungi for quinone production and evaluate their potential for future optimization. We investigated toluquinone (TQ) potentially produced by Penicillium cf. griseofulvum, terreic acid (TA) produced by Aspergillus parvulus and A. christenseniae, and anthraquinone (AQ) monomers and dimers produced by Talaromyces islandicus. The strains grew on various agar and/or liquid media and were analyzed by ultra-high-performance liquid chromatography-diode array detection-quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOF MS). In the case of AQs, feature-based molecular networking (FBMN) was used for the identification of AQ analogs. TQ was not observed in the production strains. TA constituted one of the major chromatogram peaks and was secreted into the growth medium by A. parvulus. The AQs constituted many major chromatogram peaks in the mycelium extracts and endocrocin and citreorosein were observed extracellularly in small amounts.

Dereplication of microbial natural products by LC-DAD-TOFMS.

Dereplication, the rapid identification of known compounds present in a mixture, is crucial to the fast discovery of novel natural products. Determining the elemental composition of compounds in mixtures and tentatively identifying natural products using MS/MS and UV/vis spectra is becoming easier with advances in analytical equipment and better compound databases. Here we demonstrate the use of LC-UV/vis-MS-based dereplication using data from UV/vis diode array detection and ESI+/ESI- time-of-flight MS for assignment of 719 microbial natural product and mycotoxin reference standards. ESI+ was the most versatile ionization method, detecting 93% of the compounds, although with 12% ionizing poorly. Using ESI+ alone, 56.1% of the compounds could be unambiguously assigned based on characteristic patterns of multiple adduct ions. Using ESI-, 36.4% of the compounds could have their molecular mass assigned unambiguously using multiple adduct ions, while a further 41% of the compounds were detected only as [M - H]-. The most reliable interpretations of conflicting ESI+ and ESI- data on a chromatographic peak were from the ionization polarity with the most intense ionization. Poor ionization was most common with small molecules (<200 Da). In ESI-, these were often polar and basic, while in ESI+ they were small aromatic acids or anthraquinones. No single ion-source settings could be applied over a m/z 60-2000 range. However, continuous switching among three settings (e.g., for 0.5 s each) during the chromatographic run allowed MS of both small labile molecules and large peptides, and pseudo MS/MS data on labile molecules since the settings for large molecules often induce fragmentation into small molecules.

High-yield production of hydrophobins RodA and RodB from Aspergillus fumigatus in Pichia pastoris.

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200-300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.

Salting of dry-cured meat - A potential cause of contamination with the ochratoxin A-producing species Penicillium nordicum.

Penicillium nordicum is a known contaminant of protein-rich foods and is primarily found on dry-cured meat products. It is an important producer of the mycotoxin ochratoxin A, which has nephrotoxic and cancerogenic activities. Recently a high number of P. nordicum strains was isolated from different dry-cured meat products from one of the Slovenian meat-processing plants. Since we have isolated P. nordicum in high counts also from Artic habitats, such as sea water and sea ice and due to its ability to grow well at low temperatures and at increased salinity, sea salt was suspected as the possible source of P. nordicum. In the present study contamination of meat products, air in the meat-processing plant and sea salt used for salting were analysed. When 50 g of salt sample from a sealed package was dissolved in sterile water and filtered, 12 colonies of P. nordicum were obtained on solid medium incubated at 15 °C, while a salt sample from an open vessel in the meat-processing area developed high, uncountable number of colonies. Amplified fragment length polymorphism analyses of P. nordicum isolates from different sources showed that contamination of meat products via salt was possible. Three selected isolates examined for extrolites all produced ochratoxin A. As contamination of dry-cured meat products with P. nordicum represents a potential health risk for consumers and workers in the meat-processing plants, salt should be taken into account as a potential cause of such contaminations.

The mycobiota of three dry-cured meat products from Slovenia.

The surface mycobiota of three types of Slovenian dry-cured meat products were isolated from a total of 75 items of product that were sampled periodically during the drying/ripening stage of processing. The predominant filamentous fungal genus isolated was Penicillium. Eurotium spp., Aspergillus versicolor and Cladosporium spp. were isolated from only two of the products. Eight Penicillium species were identified. Penicillium nordicum was recovered frequently. Penicillium nalgiovense was recovered less frequently, from one product only (a salami), while a yet-to-be described species Penicillium "milanense" was isolated from 21 items. The other penicillia were rarely isolated. Of the isolated and identified species, those that can produce mycotoxins are: A. versicolor, Penicillium brevicompactum, Penicillium chrysogenum, P. nordicum, and Penicillium polonicum. Their growth on dry-cured meat products is undesirable.

Growth Enhancement of Arabidopsis (Arabidopsis thaliana) and Onion (Allium cepa) With Inoculation of Three Newly Identified Mineral-Solubilizing Fungi in the Genus Aspergillus Section Nigri.

Some soil fungi play an important role in supplying elements to plants by the solubilizing of insoluble minerals in the soil. The present study was conducted to isolate the mineral-solubilizing fungi from rhizosphere soil in some agricultural areas in northern Thailand. Seven fungal strains were obtained and identified using a polyphasic taxonomic approach with multilocus phylogenetic and phenotypic (morphology and extrolite profile) analyses. All obtained fungal strains were newly identified in the genus Aspergillus section Nigri, Aspergillus chiangmaiensis (SDBR-CMUI4 and SDBR-CMU15), Aspergillus pseudopiperis (SDBR-CMUI1 and SDBR-CMUI7), and Aspergillus pseudotubingensis (SDBR-CMUO2, SDBR-CMUO8, and SDBR-CMU20). All fungal strains were able to solubilize the insoluble mineral form of calcium, copper, cobalt, iron, manganese, magnesium, zinc, phosphorus, feldspar, and kaolin in the agar plate assay. Consequently, the highest phosphate solubilization strains (SDBR-CMUI1, SDBR-CMUI4, and SDBR-CMUO2) of each fungal species were selected for evaluation of their plant growth enhancement ability on Arabidopsis and onion in laboratory and greenhouse experiments, respectively. Plant disease symptoms were not found in any treatment of fungal inoculation and control. All selected fungal strains significantly increased the leaf number, leaf length, dried biomass of shoot and root, chlorophyll content, and cellular inorganic phosphate content in both Arabidopsis and onion plants under supplementation with insoluble mineral phosphate. Additionally, the inoculation of selected fungal strains also improved the yield and quercetin content of onion bulb. Thus, the selected strains reveal the potential in plant growth promotion agents that can be applied as a biofertilizer in the future.

Discovery and Extrolite Production of Three New Species of Talaromyces Belonging to Sections Helici and Purpurei from Freshwater in Korea.

Three novel fungal species, Talaromyces gwangjuensis, T. koreana, and T. teleomorpha were found in Korea during an investigation of fungi in freshwater. The new species are described here using morphological characters, a multi-gene phylogenetic analysis of the ITS, BenA, CaM, RPB2 regions, and extrolite data. Talaromyces gwangjuensis is characterized by restricted growth on CYA, YES, monoverticillate and biverticillate conidiophores, and globose smooth-walled conidia. Talaromyces koreana is characterized by fast growth on MEA, biverticillate conidiophores, or sometimes with additional branches and the production of acid on CREA. Talaromyces teleomorpha is characterized by producing creamish-white or yellow ascomata on OA and MEA, restricted growth on CREA, and no asexual morph observed in the culture. A phylogenetic analysis of the ITS, BenA, CaM, and RPB2 sequences showed that the three new taxa form distinct monophyletic clades. Detailed descriptions, illustrations, and phylogenetic trees are provided.

Review of secondary metabolites and mycotoxins from the Aspergillus niger group.

Filamentous fungi in the Aspergillus section Nigri (the black aspergilli) represent some of the most widespread food and feed contaminants known but they are also some of the most important workhorses used by the biotechnological industry. The Nigri section consists of six commonly found species (excluding A. aculeatus and its close relatives) from which currently 145 different secondary metabolites have been isolated and/or detected. From a human and animal safety point of view, the mycotoxins ochratoxin A (from A. carbonarius and less frequently A. niger) and fumonisin B(2) (from A. niger) are currently the most problematic compounds. Especially in foods and feeds such as coffee, nuts, dried fruits, and grape-based products where fumonisin-producing fusaria are not a problem, fumonisins pose a risk. Moreover, compounds such as malformins, naptho-gamma-pyrones, and bicoumarins (kotanins) call for monitoring in food, feed, and biotechnology products as well as for a better toxicological evaluation, since they are often produced in large amounts by the black aspergilli. For chemical differentiation/identification of the less toxic species the diketopiperazine asperazine can be used as a positive marker since it is consistently produced by A. tubingensis (177 of 177 strains tested) and A. acidus (47 of 47 strains tested) but never by A. niger (140 strains tested). Naptho-gamma-pyrones are the compounds produced in the highest quantities and are produced by all six common species in the group (A. niger 134 of 140; A. tubingensis 169 of 177; A. acidus 44 of 47; A. carbonarius 40 of 40, A. brasiliensis 18 of 18; and A. ibericus three of three).

Fingerprinting using extrolite profiles and physiological data shows sub-specific groupings of Penicillium crustosum strains.

Fingerprinting of Penicillium crustosum strains was performed using different phenotypic characteristics. Seven strains of this extremely homogenous species were selected; of these, five originated from geographical locations characterized by low temperatures, and one from a location with a low water activity. Principal component analysis (PCA) was performed using micromorphological data, temperature- and water-dependent growth rates, and extrolite profiles obtained by HPLC analysis. The micromorphological data were less informative, while the growth-rate data were informative only if the strains investigated already showed slight adaptations to the selected external parameter. In contrast, PCA analyses of the extrolite data showed groupings of the strains according to their origins and known physiological differences. These groupings are in full agreement with the clustering obtained by previous amplified fragment length polymorphism (AFLP) study. We thus demonstrate here for the first time that combined qualitative and quantitative extrolite profiles can be used as a tool for phenotypic fingerprinting, to complement, or replace, molecular fingerprinting techniques.

Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001.

For a safe and sustainable environment, effective microbes as biocontrol agents are in high demand. We have isolated a new Bacillus velezensis strain DTU001, investigated its antifungal spectrum, sequenced its genome, and uncovered the production of lipopeptides in HPLC-HRMS analysis. To test the antifungal efficacy, extracts of B. velezensis DTU001 was tested against a range of twenty human or plant pathogenic fungi. We demonstrate that inhibitory potential of B. velezensis DTU001 against selected fungi is superior in comparison to single lipopeptide, either iturin or fengycin. The isolate showed analogous biofilm formation to other closely related Bacilli. To further support the biocontrol properties of the isolate, coculture with Candida albicans demonstrated that B. velezensis DTU001 exhibited excellent antiproliferation effect against C. albicans. In summary, the described isolate is a potential antifungal agent with a broad antifungal spectrum that might assist our aims to avoid hazardous pathogenic fungi and provide alternative to toxicity caused by chemicals.

Aspergillus section Flavi diversity and the role of A. novoparasiticus in aflatoxin contamination in the sugarcane production chain.

The presence of Aspergillus section Flavi and aflatoxins in sugarcane as well as in by-products, such as molasses, sugar, yeast cream and dried yeast, collected from different fields and processing plants in São Paulo state, were investigated throughout the sugarcane production chain. A total of 246 samples was collected and analyzed and 226 isolates of Aspergillus section Flavi were isolated. Aspergillus section Flavi strains were found in sugarcane juice, milled sugarcane, stalk, soil and dried yeast samples. Among the isolates of Aspergillus section Flavi submitted to polyphasic identification (n = 57), Aspergillus novoparasiticus and Aspergillus arachidicola were predominantly found. A significant proportion of the isolates (84.5%) were found to have morphological and physiological characteristics of A. novoparasiticus. Most samples, with the exception of sugar, showed some aflatoxin contamination. The highest level was in dried yeast with an average of 2.55 µg/kg and maximum value of 10.19 µg/kg. This is the first report of contamination of sugarcane by A. novoparasiticus.