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Many genes responsible for melanin biosynthesis in fungi were physically linked together. The PKS gene clusters in most of the melanin-producing fungi were regulated by the Cmr1. It was found that a close rearrangement of the PKS gene clusters had evolved in most of the melanin-producing fungi and various functions of melanin in them were beneficial to their adaptation to the changing environments. The melanin-producing fungi had undergone at least five large-scale differentiations, making their PKS gene clusters be quickly evolved and the fungi be adapted to different harsh environments. The recent gene losses and expansion were remarkably frequent in the PKS gene clusters, leading to their rapid evolution and adaptation of their hosts to different environments. The PKS gene and the CMR1 gene in them were subject to a strong co-evolution, but the horizontal gene transfer events might have occurred in the genome-duplicated species, Aspergillus and Penicillium.

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol-1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Δpks13, Δpta13, Δest1, Δp12 and Δg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Δgltp1 mutant and a Δmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.

Background The widespread use of shared bicycles has increased the demand and sanitary requirements for shared bicycles. Previous studies have identified potentially pathogenic bacteria on the surfaces of shared bicycles, but fungal communities have not been investigated. Methods We sampled shared-bicycle handles and saddles from five selected locations in a metropolis (Chengdu, China, n = 98) and used surrounding air deposition samples as controls (n = 12). Full-length ITS sequencing and multiple bioinformatic analyses were utilized to reveal fungal community structures and differences. Results Aspergillus was dominant on both the handles and saddles of shared bicycles, and Alternaria and Cladosporium were the most abundant families in the air samples. Significant differences in fungal community structures were found among the three groups. The handle samples contained higher abundances of Aureobasidium melanogenum and Filobasidium magnum than the saddle and air samples. The saddle samples had a higher abundance of Cladosporium tenuissimum than the other two sample types (P P Conclusion There were more potential pathogens, including Aspergillus pseudoglaucus, Aureobasidium melanogenum, Kazachstania pintolopesii, Filobasidium magnum, Candida tropicalis, and Malassezia globose were found on shared bicycles than in air, suggesting that hands should not contact mucous membrane after cycling, especially in susceptible individuals, and hygiene management of shared bicycles should be given more attention by relevant organizations worldwide.

Human-made hydrocarbon-rich environments are important reservoirs of microorganisms with specific degrading abilities and pathogenic potential. In particular, black fungi are of great interest, but their presence in the environment is frequently underestimated because they are difficult to isolate. In the frame of a biodiversity study from fuel-contaminated sites involving 30 diesel car tanks and 112 fuel pump dispensers (52 diesel and 60 gasoline, respectively), a total of 181 black fungal strains were isolated. The long cold incubation (LCI) of water-suspended samples, followed by plating on Dichloran Rose Bengal Chloramphenicol Agar (DRBC), gave isolation yields up to six times (6.6) higher than those of direct plating on DRBC, and those of enrichment with a phenolic mix. The sequencing of ITS and LSU-rDNA confirmed the dominance of potentially pathogenic fungi from the family Herpotrichiellaceae and Exophiala xenobiotica. Moreover, other opportunistic species were found, including E. opportunistica, E. oligosperma, E. phaeomuriformis, and Rhinocladiella similis. The recurrent presence of E. crusticola, Knufia epidermidis, Aureobasidium melanogenum, Cladosporium spp., and Scolecobasidium spp. was also recorded. Interestingly, 12% of total isolates, corresponding to 50% of taxa found (16/32), represent new species. All the novel taxa in this study were isolated by LCI. These findings suggest that black fungal diversity in hydrocarbon-rich niches remains largely unexplored and that LCI can be an efficient tool for further investigations.

Being a key industrial enzyme, tannase is extensively applied in various fields. Despite the characterizations of a large number of tannases, there are hardly a few tannases with exceptional thermostability. In this detailed study, a tannase-encoding gene named tanA was identified from Aureobasidium melanogenum T9 and heterologously expressed in Yarrowia lipolytica host of food grade. The purified tannase TanA with a molecular weight of above 63.0 kDa displayed a specific activity of 941.4 U/mg. Moreover, TanA showed optimum activity at 60°C and pH 6.0. Interestingly, TanA exhibited up to 61.3% activity after incubation for 12 h at 55°C, signifying its thermophilic property and distinguished thermostability. Additionally, TanA was a multifunctional tannase with high specific activities to catalyze the degradation of various gallic acid esters. Therefore, this study presents a novel tannase, TanA, with remarkable properties, posing as a potential candidate for food and agricultural processing.

The black yeast-like fungus Aureobasidium melanogenum is an opportunistic human pathogen frequently found indoors. Its traits, potentially linked to pathogenesis, have never been systematically studied. Here, we examine 49 A. melanogenum strains for growth at 37 °C, siderophore production, hemolytic activity, and assimilation of hydrocarbons and human neurotransmitters and report within-species variability. All but one strain grew at 37 °C. All strains produced siderophores and showed some hemolytic activity. The largest differences between strains were observed in the assimilation of hydrocarbons and human neurotransmitters. We show for the first time that fungi from the order Dothideales can assimilate aromatic hydrocarbons. To explain the background, we sequenced the genomes of all 49 strains and identified genes putatively involved in siderophore production and hemolysis. Genomic analysis revealed a fairly structured population of A.melanogenum, raising the possibility that some phylogenetic lineages have higher virulence potential than others. Population genomics indicated that the species is strictly clonal, although more than half of the genomes were diploid. The existence of relatively heterozygous diploids in an otherwise clonal species is described for only the second time in fungi. The genomic and phenotypic data from this study should help to resolve the non-trivial taxonomy of the genus Aureobasidium and reduce the medical hazards of exploiting the biotechnological potential of other, non-pathogenic species of this genus.

Since cold atmospheric pressure plasma (CAP) has not only bactericidal activity but also fungicidal activity without toxic residues and thermal damage, it is considered as an alternative method for sterilization of fungi on the surfaces of perishable foodstuffs and human bodies. Aureobasidium pullulans is a ubiquitous yeast-like fungus and called black yeast because it produces melanin, a dark biological pigment. It is well known that various melanized fungi show hyper-resistance to extreme stress conditions including high levels of radioactivity. Curiously, however, there is very little information about the fungicidal effects of CAP on melanized fungi. Therefore, we herein investigated the effects of CAP on A. pullulans, using cold atmospheric argon plasma (Ar plasma). We found that ammonium sulfate repressed the synthesis of melanin in A. pullulans as well as Aureobasidium melanogenum. Although the non-melanized A. pullulans cells were efficiently killed by the exposure of Ar plasma, the melanized cells showed the significant resistance to Ar plasma as well as to hydrogen peroxide and thermal stress. In order to improve the fungicidal efficacy of Ar plasma, we examined the combination of Ar plasma and Fenton reaction. We realized that FeCl2 and FeSO4 significantly improved the sterilization efficacy of Ar plasma on the melanized A. pullulans.

Aureobasidium pullulans and A. melanogenum are black-yeast-like surface colonisers and are commonly encountered as contaminants in the hospital. The species are able to produce melanin which play a role in protection against environmental stress and irradiation. Aureobasidium melanogenum shows higher frequency in opportunistic infections compared to A. pullulans. Comparative pathogenicity of opportunistic black yeasts between Aureobasidium pullulans and A. melanogenum to explain the observed differences in frequency in infection. Degrees of melanisation and thermotolerance were measured, and virulence of strains from different sources was examined in Galleria mellonela and murine infection models. Aureobasidium melanogenum responds with increased melanisation to temperature stress and generally survives at 37°C, A. pullulans on average scored less on these parameters. In the murine model, differences between species were not significant, but the melanised A. melanogenum group showed the highest virulence. This result was not reproducible in Galleria mellonella larvae at 25°C. The A. melanogenum black group showed higher pathogenicity in murine model, indicating that the combination of melanisation and thermotolerance rather than species affiliation is instrumental. Galleria larvae did not survive very well at 37°C, and hence, this model is judged insufficient to detect the small virulence differences observed in Aureobasidium.

Aureobasidium melanogenum has been used as an animal feed additive for improving thehealth of pets, however, it has not yet been applied in honey bees. Here, a fungal strain CK-CsC isolated from bee bread pollen, was identified as A. melanogenum. Following characterizing CK-CsC fermentation broth, the 4-days fermentation broth (SYM medium or bee pollen) of the CK-CsC was used to feed newly emerged adult honey bees in cages under laboratory-controlled conditions for analysis of survival, gene expression of nutrient and antibacterial peptide, and gut microbiota of honey bees. It was found that the CK–CsC fermentation broth (SYM medium or bee pollen) is nontoxic to honey bees, and can regularly increase nutrient gene expression of honey bees. However, significant mortality of bees was observed after bees were fed on the supernatant liquid of the fermentation broth. Notably, this mortality can be lowered by the simultaneous consumption of bee pollen. The honey bees that were fed bee pollen exhibited more γ-Proteobacteria, Bacteriodetes, and Actinobacteria in their gut flora than did the honey bees fed only crude supernatant liquid extract. These findings indicate that A. melanogenum CK–CsC has high potential as a bee probiotic when it was fermented with bee pollen.

Summary Currently, piperazic acid is chemically synthesized using ecologically unfriendly processes. Microbial synthesis from glucose is an attractive alternative to chemical synthesis. In this study, we report the production of L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum; this strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring and constitutive genetic circuit reconstitution, to build a robust L‐piperazic acid synthetic cascade. These genetic modifications enable A. melanogenum to directly convert glucose to L‐piperazic acid without relying on the use of either chemically synthesized precursors or harsh conditions. This bio‐based process overcomes the shortcomings of the conventional synthesis routes. The ultimately engineered strain is a very high‐efficient cell factory that can excrete 1.12 ± 0.05 g l‐1 of L‐piperazic acid after a 120‐h 10.0‐l fed‐batch fermentation; this is the highest titre of L‐piperazic acid reported using a microbial cell factory., L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum was achieved. This strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring, and constitutive genetic circuit reconstitution to build a robust L‐piperazic acid synthetic cascade. The ultimately engineered strain has the highest titer of L‐piperazic acid of 1.12 ± 0.05 g l‐1 ever reported.

The pathogenic yeast strain LIAO causing the milky disease in the Chinese mitten crab belonged to one member of Metschnikowia bicuspidate which could grow well at different temperatures from 28 to 4 °C. It was also found that the pathogenic yeast strain LIAO could grow in the extracts of the muscle, gill, heart tissues, intestinal tracts of the healthy Chinese mitten crabs by using the reducing sugars, amino acids and other nutrients in them. Massoia lactone released from liamocins produced by Aureobasidium melanogenum had high anti-fungal activity against the pathogenic yeast strain LIAO and M. bicuspidate WCY isolated from the diseased marine crabs. The minimal inhibitory concentrations (MIC) and the minimal fungicidal concentration (MFC) in the liquid culture against the pathogenic yeast strain LIAO were 0.15 mg/mL and 0.34 mg/mL, respectively. Massoia lactone as a bio-surfactant could damage the cell membrane, even break the whole cells of the pathogenic yeast strain LIAO and cause cellular necrosis of the pathogenic yeast LIAO. Therefore, Massoia lactone could be used to effectively kill the pathogenic yeast strains and as an effectitve treatment for milky disease in the Chinese mitten crab.

Some soil microorganisms, especially bacteria and mycorrhizal fungi, play a role in the promotion of plant growth. However, plant growth promotion involving yeasts in soil has not yet been extensively investigated. This study aimed to isolate and identify yeast strains obtained from soils of the Assam tea plant (Camellia sinensis var. assamica) in northern Thailand and to investigate their plant growth promoting capabilities. A total of 42 yeast strains were obtained and identified by analysis of the D1/D2 domain of the large subunit ribosomal RNA gene. We identified 35 strains of six species belonging to the phylum Ascomycota, namely Aureobasidium melanogenum, Kazachstania aquatica, Saturnispora diversa, Saturnispora sekii, Schwanniomyces pseudopolymorphus and Wickerhamomyces anomalus, and six species were determined to belong to the phylum Basidiomycota, namely Apiotrichum scarabaeorum, Curvibasidium pallidicorallinum, Papiliotrema laurentii, Rhodosporidiobolus ruineniae, Trichosporon asahii and Trichosporon coremiiforme. Seven strains were representative of potential new species and belonged to the genera Galactomyces and Wickerhamomyces. A total of 28 strains were found to produce indole-3-acetic acid (IAA) in a range of 2.12 to 37.32 mg/L, with the highest amount of IAA produced by R. ruineniae SDBR-CMU-S1-03. All yeast strains were positive in terms of ammonia production, and only eight strains were positive for siderophore production. Two yeast species, P. laurentii and W. anomalus, were able to solubilize the insoluble form of calcium and zinc. The ability to produce amylase, endogulcanase, lipase, pectinase, protease and xylanase was dependent upon the yeast species and strain involved.

There has been an increase in the occurrence of fungal diseases in Ilex paraguariensis A. St.-Hill. (yerba mate) resulting from their domestication. Seed health tests may indicate pathogens prior to sowing, enabling their control. Our study sought to compare the efficacy of fungi detection methods on yerba mate seeds. Seeds from four lots were subjected to the following detection methods: BDA, V8 and filter paper; as well as the performance of asepsis with 1% sodium hypochlorite and 70% alcohol. The fungi found in the lots were: Penicillium spp., Cladosporium spp., Aureobasidium melanogenum, Aspergillus spp., Pestalotia spp., Nigrospora spp., Trichoderma spp., Fusarium graminearum, F. oxysporum and F. guttiforme. The asepsis in the seeds reduced the occurrence of contaminating organisms. There were differences between the evaluated lots regarding fungal incidence. In general, the BDA and V8 culture media showed better results in fungal detection.

The identification of yeasts or yeasts-like fungi and verify their diversity are principal aspect for bioindustry and ecosystem sustainability. Taxonomic approach provides identification tool to ensure the taxonomic position of yeasts and yeasts-like fungi which definitely set to utilization concerns. The aim of this study is to understanding the taxonomic position of yeasts and yeasts-like fungi from the distinctive of its sequences relationship. Yeasts and yeasts-like fungi strains were isolated through various culture dependent methods from natural resources samples of Karimun Besar Island, Province of Riau Islands, Indonesia. The identification process was performed through amplifying the accurate DNA-based in D1/D2 region of large subunit (26S) ribosomal DNA. As the result, a total of 85 isolates of yeasts and yeasts-like fungi were obtained with 16 closest related taxa through phylogenetic tree construction. Ascomycetous was the predominating group representing 91% of the total isolates sequences followed by Basidiomycetous (8%) and Zygomycetous (1%). The black yeasts (yeasts-like) known as Aureobasidium melanogenum was predominant species with represent to 54% of total isolates and present in particular habitat. Taxonomically, there are six isolates are represent to be novel taxa candidates which pretend to enhance genetic resources of yeasts and yeasts-like fungi especially from Indonesia. In addition, this information provides specific technique to reach specific yeasts or yeasts-like fungi species in nature by managing the sample collection and culture methods.

A PKS1 gene responsible for the melanin biosynthesis and a NPG1 gene in Aureobasidium melanogenum XJ5-1 were cloned and characterized. An ORF of the PKS1 gene encoding a protein with 2165 amino acids contained 6495bp while an ORF of the NPG1 gene encoding a protein with 340 amino acids had 1076bp. After analysis of their promoters, it was found that expression of both the PKS1 gene and the NPG1 gene was repressed by nitrogen sources and glucose, respectively. The PKS deduced from the cloned gene consisted of one ketosynthase, one acyl transferase, two acyl carrier proteins, one thioesterase and one cyclase while the PPTase belonged to the family Sfp-type. After disruption of the PKS1 gene and the NPG1 gene, expression of the PKS1 gene and the NPG1 gene and the melanin biosynthesis in the disruptants K5 and DP107 disappeared and expression of the PKS1 gene in the disruptant DP107 was also negatively influenced. However, after the NPG1 gene was complemented in the disruptant DP107, the melanin biosynthesis in the complementary strain BP17 was restored and expression of the PKS1 gene and the NPG1 gene was greatly enhanced, suggesting that the PKS was indeed activated and regulated by the PPTase and expression of the PKS1 gene and the NPG1 gene had a coordinate regulation.

Endophytic fungi are fungi which live inside the host plant tissue and have been undergone a horizontal gene transfer process. Endophytic fungi are able to synthesize the same bioactive compounds which synthesized by their host plants. The host plant used in this research was dragon’s scales fern (Pyrrosia piloselloides (L.) M.G. Price). Dragon’s scales fern produces various of bioactive compounds which used as antibacterial agents such as polyphenols. This research was aimed to obtain endophytic fungi isolates from trophophyll fronds and sporophyll fronds of dragon’s scales fern, to determine the antibacterial activity of the secondary metabolite extracts of endophytic fungi, to determine the Minimum Inhibitory Concentration (MIC), to determine the characteristics of the endophytic fungi isolates which potentially as antibacterial source. Based on the research, 13 endophytic fungi isolates were obtained from dragon’s scales fern fronds consist of 5 isolates from trophophyll fronds and 8 isolates from sporophyll fronds. The antibacterial activity test showed that the extract of secondary metabolites of the isolate DTP2 had the highest inhibition zone diameter against E.coli 14.82 ± 4.05 mm, DTP4 against S.aureus 8.80 ± 0.03 mm and DSP4 against S.dysentriae 10.15 ± 0.36 mm. MIC of ethyl acetate extracts of secondary metabolites of isolate DTP2 against E.coli was 125 µg/mL, DTP4 against S.aureus was 125 µg/mL and DSP4 against S.dysentriae was 31.25 µg/mL. The endophytic fungi isolate DTP2 identified as Aureobasidium melanogenum, DTP4 identified as Penicillium alliisativi and DSP4 identified as Aspergillus flocculosus.

Xylose, the main component of xylan, is the second most abundant sugar in nature after glucose. Consequently, xylose represents an attractive feedstock for the production of value-added compounds such as biosurfactants (BSs), which are produced by various bacteria and yeasts. In this study, we screened and isolated yeast strains that synthesize BSs using xylose as the sole carbon source. We applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to screen for BS-producing yeasts and isolated eight strains as the liamocin producers. Two of the eight strains, AS37 and SK25, were identified as Aureobasidium melanogenum, which is known as black yeasts, by based on 26S ribosomal RNA gene sequences. Both strains produced a wide variety of liamocin structures from not only xylose but also glucose and sucrose. According to the MALDI-TOF MS analysis, signals corresponding to sodium ion adducts of di-, tri-, tetra-, penta- and hexa-acylated C6-liamocins and di-, tri- and tetra-acylated C5-liamocins were detected. In addition, their mono-acetylated form was also detected. The dominant sugar component of liamocins produced by strains AS37 and SK25 is mannitol as estimated by HPLC analysis. This is the first report to describe the screening of liamocins-producing yeasts using xylose as the sole carbon source.

The maximum yield of xylanase from Aureobasidium melanogenum PBUAP46 was 5.19 ± 0.08 U ml−1 when cultured in a production medium containing 3.89% (w/v) rice straw and 0.75% (w/v) NaNO3 as carbon and nitrogen sources, respectively, for 72 h. This enzyme catalyzed well and was relatively stable at pH 7.0 and room temperature (28 ± 2 °C). The produced xylanase was used to hydrolyze xylans from four tropical weeds, whereupon it was found that the highest amounts of reducing sugars in the xylan hydrolysates of cogon grass (Imperata cylindrical), Napier grass (Pennisetum purpureum), and vetiver grass (Vetiveria zizanioides) were at 20.44 ± 0.84, 17.50 ± 0.29, and 19.44 ± 0.40 mg 100 mg xylan−1, respectively, but it was not detectable in water hyacinth (Eichhornia crassipes) hydrolysate. The highest combined amount of xylobiose and xylotriose was obtained from vetiver grass; thus, it was selected for further optimization. After optimization, xylanase digestion of vetiver grass xylan at 27.94 U g xylan−1 for 92 h 19 min gave the highest amount of reducing sugars (23.65 ± 1.34 mg 100 mg xylan−1), which were principally xylobiose and xylotriose. The enriched XOs exhibited a prebiotic property, significantly stimulating the growth of Lactobacillus brevis and L. casei by a factor of up to 3.5- and 6.5-fold, respectively, compared to glucose.

Liamocins, as the secondary metabolites synthesized and secreted by Aureobasidium spp., consist of a single mannitol or a single arabitol head group partially O-acylated with three 3,5-dihydroxydecanoic ester groups or directly esterified with three or four 3,5-dihydroxydecanoic ester tails. Very recently, the whole synthetic pathway of liamocins in A. melanogenum 6-1-2 has been elucidated. It was found that the promoter sequences of all the genes related to liamocin synthesis in A. melanogenum 6-1-2 had stress regulatory elements with core sequences of AGGGG or CCCCT. Therefore, expression of all the genes would be regulated by the Msn2. In this study, it was found that removal of the single one MSN2 gene in A. melanogenum 6-1-2 made the mutant decrease yield of extracellular liamocin by 92.28 %, while complementation of the MSN2 gene in the mutant rendered liamocin synthesis to be restored. When A. melanogenum 6-1-2 was cultured in the liamocin fermentation medium with high glucose and low nitrogen, the Msn2 was localized in the nucleus and positively regulated the expression of the genes related to liamocin biosynthesis. Furthermore, when the key BCY1 gene encoding regulatory subunit of the cAMP-PKA signaling pathway in A. melanogenum 6-1-2 was knocked out, the amount of extracellular liamocins synthesized by the mutant was decreased by 96.73 % and the Msn2 was localized in the cytoplasm. Similarly, when the key HOG1 gene in the HOG1 signaling pathway was deleted, liamocin biosynthesis in the knockout strain was decreased by 98.09 %. However, it was found that the Hog1 may be one part of the general transcription complex to regulate the transcription of the MSN2 gene, leading to the reduced Msn2 and liamocin synthesis in the mutant. In addition, the key TOR1 gene and SNF1 gene in the TOR1 signaling pathway and the SNF1 signaling pathway were not involved in the regulation of the Msn2 activity and liamocin synthesis. It was concluded that the transcriptional activator Msn2, the HOG1 signaling pathway and the cAMP-PKA signaling pathway were involved in the regulation of liamocin biosynthesis and production.

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg2+, Mn2+, Li+, Ca2+, Ni2+, CHAPS, DTT, and EDTA and inhibited by Hg2+, Ag+, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.

Cost-efficient production of pullulan is of great importance but remains challenging due largely to the high-cost carbon sources. Lignocellulosic biomass is considered an alternative carbon source for industrial pullulan production, while new fungus producers that co-utilizing lignocellulose-derived glucose and xylose are required. In this study, a new fungus Aureobasidium melanogenum TN2-1-2 showed simultaneously assimilation of glucose and xylose and could produce pigment-free pullulan due to its deficiency in melanin synthesis. The ability of TN2-1-2 producing pullulan was remarkably robust in the presence of varying glucose to xylose ratios and ionic salt concentrations. Furthermore, condensed lignocellulosic hydrolysates obtained by consolidated biosaccharification was used as the pullulan production medium without supplying any nutrients, and pigment-free pullulan was produced by TN2-1-2 with the titer and yield of 55.1 g/L and 0.5 gPullulan/gCBS hydrolysate, respectively. Hence, this work provides a potential industrial pullulan producer TN2-1-2 and new insight into the lignocellulose bioconversion to pullulan.

Background/aim Oxidative stress caused by the production of excessive cellular reactive oxygen species (ROS) and high levels of nitric oxide contribute to several human pathologies. This study aimed to examine the anti-oxidant effects of fusigen, a compound produced from Aureobasidium melanogenum. Materials and methods Extracts of A. melanogenum were selected as a source for the isolation of fusigen. The anti-oxidant, nitric oxide suppression, as well as the free radical scavenging activities of fusigen were tested in BEAS-2B human bronchial epithelial cell line (BEAS-2B cells) and human dermal papilla cells (DP cells) using specific fluorescence dyes and flow cytometry analysis. Cell viability was determined by the MTT assay. Results Fusigen did not exert cytotoxicity in the human normal BEAS-2B and DP cells at concentrations up to 100 μM. Fusigen decreased basal levels of cellular ROS, as well as the levels of ROS induced by hydrogen peroxide and ferrous ion enrichment. ROS decreasing effect was confirmed in DP cells. In addition, fusigen treatment suppressed intracellular NO levels in both BEAS-2B and DP cells. Conclusion The optimal process of production of purified fusigen from A. melanogenum was determined. Fusigen exhibited a low cytotoxic effect and the potential to suppress ROS and NO. These results demonstrated that fusigen may be used for the treatment or prevention of human diseases.

Dark homogenous fungal‐based layers called biofinishes and vegetable oils are key ingredients of an innovative wood protecting system. The aim of this study was to determine which of the vegetable oils that have been used to generate biofinishes on wood will provide carbon and energy for the biofinish‐inhabiting fungus Aureobasidium melanogenum, and to determine the effect of the oil type and the amount of oil on the cell yield. Aureobasidium melanogenum was cultivated in shake flasks with different types and amounts of carbon‐based nutrients. Oil‐related total cell and colony‐forming unit growth were demonstrated in suspensions with initially 1% raw linseed, stand linseed, and olive oil. Oil‐related cell growth was also demonstrated with raw linseed oil, using an initial amount of 0.02% and an oil addition during cultivation. Nile red staining showed the accumulation of fatty acids inside cells grown in the presence of oil. In conclusion, each tested vegetable oil was used as carbon and energy source by A. melanogenum. The results indicated that stand linseed oil provides less carbon and energy than olive and raw linseed oil. This research is a fundamental step in unraveling the effects of vegetable oils on biofinish formation.

Stress-tolerant fungi that can thrive under various environmental extremes are highly desirable for their application to biological control, as an alternative to chemicals for pest management. However, in fungi, the mechanisms of stress tolerance might also have roles in mammal opportunism. We tested five species with high biocontrol potential in agriculture (Aureobasidium pullulans, Debayomyces hansenii, Meyerozyma guilliermondii, Metschnikowia fructicola, Rhodotorula mucilaginosa) and two species recognized as emerging opportunistic human pathogens (Exophiala dermatitidis, Aureobasidium melanogenum) for growth under oligotrophic conditions and at 37 °C, and for tolerance to oxidative stress, formation of biofilms, production of hydrolytic enzymes and siderophores, and use of hydrocarbons as sole carbon source. The results show large overlap between traits desirable for biocontrol and traits linked to opportunism (growth under oligotrophic conditions, production of siderophores, high oxidative stress tolerance, and specific enzyme activities). Based on existing knowledge and these data, we suggest that oligotrophism and thermotolerance together with siderophore production at 37 °C, urease activity, melanization, and biofilm production are the main traits that increase the potential for fungi to cause opportunistic infections in mammals. These traits should be carefully considered when assessing safety of potential biocontrol agents.

Aureobasidium melanogenum HN6.2 is a unique yeast strain who can produce the siderophore of fusigen under iron starvation to guarantee its survival. However, a comprehensive understanding of mechanisms involved in iron acquisition and homeostasis for it is still vacant. In this study, genome sequencing and mining revealed that A. melanogenum HN6.2 strain was the first yeast species that exclusively possessed all the four known mechanisms for the iron acquisition: (i) the siderophore-mediated iron uptake; (ii) reductive iron assimilation; (iii) low-affinity ferrous uptake; and (iv) heme utilization, which suggested its stronger adaptability than Aspergillus fumigatus and Saccharomyces cerevisiae. This HN6.2 strain also employed the vacuolar iron storage for immobilizing the excessive iron to avoid its cellular toxicity. Specially, genome mining indicated that A. melanogenum HN6.2 strain could also synthesize ferricrocin siderophore. Further HPLC and Q-Tof-MS analysis confirmed that the siderophores synthesized by this strain consisted of cyclic fusigen, linear fusigen, ferricrocin, and hydroxyferricrocin and they played parallel roles as both intracellular and extracellular siderophores. Also, the heme utilization for this strain was experimentally verified by the knock-out of heme oxygenase gene. For iron homeostasis, the transcriptome analysis revealed that this strain mainly employed two central regulators of SreA/HapX to tune iron uptake and storage at the transcriptional level. It was also noted that mitogen-activated protein kinase C gene (MpkC) exhibited a transcriptional up-regulation under iron sufficiency, suggesting that it may serve as another factor involved in the repression of siderophore biosynthesis. This is the first genetic blueprint of iron acquisition and homeostasis for A. melanogenum.

Around 85% of the environments on Earth are permanently or seasonally colder than 5 &deg, C. Among those, the poles constitute unique biomes, which harbor a broad variety of microbial life, including an abundance of fungi. Many fungi have an outstanding ability to withstand extreme conditions and play vital ecosystem roles of decomposers as well as obligate or facultative symbionts of many other organisms. Due to their dispersal capabilities, microorganisms from cryosphere samples can be distributed around the world. Such dispersal involves both species with undefined pathogenicity and potentially pathogenic strains. Here we describe the isolation of fungal species from pristine Arctic locations in Greenland and Svalbard and the testing of the expression of characteristics usually associated with pathogenic species, such as growth at 37 &deg, C, hemolytic ability, and susceptibility to antifungal agents. A total of 320 fungal isolates were obtained, and 24 of the most abundant and representative species were further analyzed. Species known as emerging pathogens, like Aureobasidium melanogenum, Naganishia albida, and Rhodotorula mucilaginosa, were able to grow at 37 &deg, C, showed beta-hemolytic activity, and were intrinsically resistant to commonly used antifungals such as azoles and echinocandins. Antifungal resistance screening revealed a low susceptibility to voriconazole in N. albida and Penicillium spp. and to fluconazole in Glaciozyma watsonii and Glaciozyma-related taxon.

The genus Aureobasidium, which is known as a wood staining mould, has been detected on oil treated woods in the specific stain formation called biofinish. This biofinish is used to develop a new protective, self-healing and decorative biotreatment for wood. In order to understand and control biofinish formation on oil treated wood, the occurrence of different Aureobasidium species on various wood surfaces was studied. Phenotypic variability within Aureobasidium strains presented limitations of morphological identification of Aureobasidium species. PCR amplification and Sanger sequencing of ITS and RPB2 were used to identify the culturable Aureobasidium species composition in mould stained wood surfaces with and without a biofinish. The analysed isolates showed that several Aureobasidium species were present and that Aureobasidium melanogenum was predominantly detected, regardless of the presence of a biofinish and the type of substrate. A.melanogenum was detected on wood samples exposed in the Netherlands, Cameroon, South Africa, Australia and Norway. ITS-specific PCR amplification, cloning and sequencing of DNA extracted from biofinish samples confirmed results of the culturing based method: A. melanogenum is predominant within the Aureobasidium population of biofinishes on pine sapwood treated with raw linseed oil and the outdoor placement in the Netherlands. Electronic supplementary material The online version of this article (doi:10.1007/s10482-016-0668-7) contains supplementary material, which is available to authorized users.