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.

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.

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.

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.

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.

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.

Uncovering secondary metabolite evolution and biosynthesis using gene cluster networks and genetic dereplication.

The increased interest in secondary metabolites (SMs) has driven a number of genome sequencing projects to elucidate their biosynthetic pathways. As a result, studies revealed that the number of secondary metabolite gene clusters (SMGCs) greatly outnumbers detected compounds, challenging current methods to dereplicate and categorize this amount of gene clusters on a larger scale. Here, we present an automated workflow for the genetic dereplication and analysis of secondary metabolism genes in fungi. Focusing on the secondary metabolite rich genus Aspergillus, we categorize SMGCs across genomes into SMGC families using network analysis. Our method elucidates the diversity and dynamics of secondary metabolism in section Nigri, showing that SMGC diversity within the section has the same magnitude as within the genus. Using our genome analysis we were able to predict the gene cluster responsible for biosynthesis of malformin, a potentiator of anti-cancer drugs, in 18 strains. To proof the general validity of our predictions, we developed genetic engineering tools in Aspergillus brasiliensis and subsequently verified the genes for biosynthesis of malformin.

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.

Diversity of Aspergillus section Nigri on the surface of Vitis labrusca and its hybrid grapes.

This study investigated the presence of Aspergillus species belonging to Aspergillus section Nigri on Vitis labrusca and its hybrid grapes grown in Brazil. The ability of the fungi isolates to produce ochratoxin A (OTA) and fumonisin B2 (FB2) as well as the presence of these mycotoxins in the grapes were also studied. Eighty-eight samples were collected from the main grape producing states in Brazil: Rio Grande do Sul (n=30), Pernambuco (n=21), São Paulo (n=21) and Paraná (n=16). The highest average contamination level by A. section Nigri occurred on the grapes from Pernambuco (66.3%). A total of 2042 A. section Nigri isolates was analyzed and clustered in three groups according to morphology characterization: A. section Nigri uniseriate (79.3%), A. niger "aggregate" (18.3%) and A. carbonarius (2.4%). In order to precisely identify the Aspergillus species, two hundred and forty-eight strains were subjected to DNA sequencing. Among the A. section Nigri uniseriate group, the following species were found: A. japonicus, A. uvarum, A. brunneoviolaceus, A. aculeatus and A. labruscus. Within the A. niger "aggregate", the following species were found: A.niger sensu stricto, A. welwitschiae and A. vadensis. Regarding mycotoxin-production capacity, 3.2% of the total A. section Nigri isolates (2042) were positive for OTA production and from A. niger "aggregate" (373) tested, 42.1% were FB2 producers. However, none of the 88 grape samples were contaminated with these mycotoxins.

Physiological characterization of secondary metabolite producing Penicillium cell factories.

BACKGROUND: Penicillium species are important producers of bioactive secondary metabolites. However, the immense diversity of the fungal kingdom is only scarcely represented in industrial bioprocesses and the upscaling of compound production remains a costly and labor intensive challenge. In order to facilitate the development of novel secondary metabolite producing processes, two routes are typically explored: optimization of the native producer or transferring the enzymatic pathway into a heterologous host. Recent genome sequencing of ten Penicillium species showed the vast amount of secondary metabolite gene clusters present in their genomes, and makes them accessible for rational strain improvement. In this study, we aimed to characterize the potential of these ten Penicillium species as native producing cell factories by testing their growth performance and secondary metabolite production in submerged cultivations. RESULTS: Cultivation of the fungal species in controlled submerged bioreactors showed that the ten wild type Penicillium species had promising, highly reproducible growth characteristics in two different media. Analysis of the secondary metabolite production using liquid chromatography coupled with high resolution mass spectrometry proved that the species produced a broad range of secondary metabolites, at different stages of the fermentations. Metabolite profiling for identification of the known compounds resulted in identification of 34 metabolites; which included several with bioactive properties such as antibacterial, antifungal and anti-cancer activities. Additionally, several novel species-metabolite relationships were found. CONCLUSIONS: This study demonstrates that the fermentation characteristics and the highly reproducible performance in bioreactors of ten recently genome sequenced Penicillium species should be considered as very encouraging for the application of native hosts for production via submerged fermentation. The results are particularly promising for the potential development of the ten analysed Penicillium species for production of novel bioactive compounds via submerged fermentations.

Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species.

Filamentous fungi produce a wide range of bioactive compounds with important pharmaceutical applications, such as antibiotic penicillins and cholesterol-lowering statins. However, less attention has been paid to fungal secondary metabolites compared to those from bacteria. In this study, we sequenced the genomes of 9 Penicillium species and, together with 15 published genomes, we investigated the secondary metabolism of Penicillium and identified an immense, unexploited potential for producing secondary metabolites by this genus. A total of 1,317 putative biosynthetic gene clusters (BGCs) were identified, and polyketide synthase and non-ribosomal peptide synthetase based BGCs were grouped into gene cluster families and mapped to known pathways. The grouping of BGCs allowed us to study the evolutionary trajectory of pathways based on 6-methylsalicylic acid (6-MSA) synthases. Finally, we cross-referenced the predicted pathways with published data on the production of secondary metabolites and experimentally validated the production of antibiotic yanuthones in Penicillia and identified a previously undescribed compound from the yanuthone pathway. This study is the first genus-wide analysis of the genomic diversity of Penicillia and highlights the potential of these species as a source of new antibiotics and other pharmaceuticals.

The biodiversity of Aspergillus section Flavi and aflatoxins in the Brazilian peanut production chain.

A total of 119 samples of peanut were collected throughout the peanut production chain in São Paulo State, Brazil. The peanut samples were directly plated for determination of percentages of infection and a polyphasic approach was used to identify Aspergillus section Flavi species. Further, the potential for aflatoxin production by the isolates was tested using the agar plug technique and the presence of aflatoxins in peanuts was assessed using an immunoaffinity column followed by quantification using HPLC with reverse phase column and fluorescence detection. The limit of detection and quantification were 0.05 and 0.17µg/kg for total aflatoxins, respectively. Four species of Aspergillus section Flavi were isolated: A. caelatus (11), A. flavus (515), A. parasiticus (17) and A. tamarii (13). All isolates of A. parasiticus were able to produce aflatoxin B and G whereas aflatoxin B was produced by 50% of A. flavus isolates. Aflatoxins were found in 12 samples at concentrations ranging from 0.3 to 100µg/kg. The data reported in this study add information on the occurrence and biodiversity of fungi in peanuts at several stages of the production chain. The occurrence of aflatoxins is also of major relevance for continuous monitoring and assessment of likely exposure of consumers to aflatoxins through consumption of peanuts.

Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of ß-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.

Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites.

A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311T = IBT 12289T). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted in the identification of 62 putative biosynthetic gene clusters. Extracts of P. arizonense were analysed for secondary metabolites and austalides, pyripyropenes, tryptoquivalines, fumagillin, pseurotin A, curvulinic acid and xanthoepocin were detected. A comparative analysis against known pathways enabled the proposal of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential industrial applications for the new species P. arizonense. The description and availability of the genome sequence of P. arizonense, further provides the basis for biotechnological exploitation of this species.

Production of the Fusarium Mycotoxin Moniliformin by Penicillium melanoconidium.

Moniliformin is a mycotoxin produced by several cereal associated Fusaria. Here, we show for the first time that moniliformin can be produced by the cereal fungus, Penicillium melanoconidium (4 out of 4 strains), but not in the related species in the Viridicata series. Moniliformin was detected in 10 out of 11 media: two agars and several cereal and bean types. Moniliformin was identified by a novel mixed-mode anionic exchange reversed phase chromatographic method which was coupled to both tandem mass spectrometry (MS) and high resolution MS. Mixed-mode chromatography showed superior peak shape compared to that of HILIC and less matrix interference compared to that of reversed phase chromatography, but during a large series of analyses, the column was fouled by matrix interferences. Wheat and beans were artificially infected by P. melanoconidium containing up to 64 and 11 mg/kg moniliformin, respectively, while penicillic acid, roquefortine C, and penitrem A levels in wheat were up to 1095, 38, and 119 mg/kg, respectively.

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.

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.

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.