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This review explores the production of the fungal polysaccharide pullulan by mutants and natural isolates of Aureobasidium species using strain improvement. Pullulan is a neutral polysaccharide gum whose structure is a maltotriose-containing glucan. This polysaccharide gum has applications in the fields of food, pharmaceuticals, biomedical and wastewater treatment. The strain improvement of Aureobasidium species has focused on the pullulan production process, including the isolation of strains exhibiting reduced pigmentation, polysaccharide overproduction, the production of pullulan with variable molecular weight, and increased osmotolerant strains promoting pullulan production at high carbon source concentrations and pullulan production on hemicellulosic substrates. The majority of studies have emphasized the isolation of reduced pigmentation and pullulan hyperproducer strains since the goal of large-scale commercial pullulan production is to synthesize non-pigmented polysaccharides. A promising area of strain improvement is the isolation of strains that synthesize authentic pullulan from hemicellulosic substrates. If strain improvement in this area is successful, the goal of commercially producing pullulan at a competitive cost will eventually be achieved. [ABSTRACT FROM AUTHOR]

Natural melanin is a biopolymer with wide application prospects in medicine, food, cosmetics, environmental protection, agriculture, and so on. Microbial fermentation is an important and effective way to produce melanin. In this study, Aureobasidium melanogenum, known as black yeast with cellular pleomorphism, was used for the production of melanin. Based on the characteristic of A. melanogenum secreting melanin under oligotrophic stress, a simple medium containing only glucose, MgSO4·7H2O, and KCl was constructed for the production of melanin. The melanin titer of 6.64 ± 0.22 g/L was obtained after 20 days of fermentation without pH control. The cell morphological changes of A. melanogenum during the production of melanin were recorded, and the results showed that chlamydospore might be the most favorable cell morphology for melanin synthesis. Then, different fermentation strategies with cell morphology analysis were developed to further improve the production of melanin in a 5-L fermenter. Results showed that the maximum titer of melanin reached 18.50 g/L by using the fermentation strategy integrating pH control, ammonium salt addition, and H2O2 stimulation, which increased by 178.6% than that of the strategy without pH control. Furthermore, the melanin obtained from the fermentation broth was characterized as eumelanin containing an indole structure. This study provided a potentially feasible fermentation strategy for the industrial production of melanin. [ABSTRACT FROM AUTHOR]

Pullulan, which is a microbial exopolysaccharide, has found widespread applications in foods, biomedicines, and cosmetics. Despite its versatility, most wild-type strains tend to yield low levels of pullulan production, and their mutants present genetic instability, achieving a limited increase in pullulan production. Therefore, mining new wild strains with robust pullulan-producing abilities remains an urgent concern. In this study, we found a novel strain, namely, Aureobasidium melanogenum ZH27, that had a remarkable pullulan-producing capacity and optimized its cultivation conditions using the one-factor-at-a-time method. To elucidate the reasons that drove the hyper-production of pullulan, we scrutinized changes in cell morphology and gene expressions. The results reveal that strain ZH27 achieved 115.4 ± 1.82 g/L pullulan with a productivity of 0.87 g/L/h during batch fermentation within 132 h under the optimized condition (OC). This pullulan titer increased by 105% compared with the initial condition (IC). Intriguingly, under the OC, swollen cells featuring 1–2 large vacuoles predominated during a rapid pullulan accumulation, while these swollen cells with one large vacuole and several smaller ones were prevalent under the IC. Moreover, the expressions of genes associated with pullulan accumulation and by-product synthesis were almost all upregulated. These findings suggest that swollen cells and large vacuoles may play pivotal roles in the high level of pullulan production, and the accumulation of by-products also potentially contributes to pullulan synthesis. This study provides a novel and promising candidate for industrial pullulan production. [ABSTRACT FROM AUTHOR]

In this study, we applied PacBio Sequel II and Illumina NovaSeq 6000 sequencing platforms to sequence the genome of a high-polymalic acid (PMLA)-producing strain, Aureobasidium melanogenum CGMCC18996, and used different assemblers to obtain a high-quality genome assembly, which was then annotated using transcriptomic data. The results indicated a total of 6 202 genes were found in the A. melanogenum genome, mainly involved in carbohydrate transport and metabolism, amino acid transport and metabolism, post-translational modification, RNA processing and modification. Meanwhile, functional annotation revealed that most genes in the genome were related to peroxisome in the strain. Transmission electron microscopy (TEM) indicated the existence of a circular peroxisome-like (glyoxysome) structure in the cells, demonstrating the ability to malic acid through the glyoxylate cycle. Finally, we predicted the protein structures of two enzymes related to PMLA biosynthesis, phosphoenolpyruvate carboxykinase (PCKA) and malate synthase (MASY). It was found that the enzymes could have the ability to synthesize malic acid. This study could provide a reference for metabolism regulation in A. melanogenum for improved PMLA production, and the assembled genome has been uploaded to the database, which could provide the basis for the future development and utilization of A. melanogenum CGMCC18996.

β-Poly(L-malic acid) (PMLA) is a water-soluble biopolymer used in food, medicine and other industries. To date, the biosynthesis pathway of PMLA has not been fully elucidated. In this study, we sequenced the transcriptome of strain Aureobasidium melanogenum under 20 g/L CaCO3 addition. The resulting sequencing reads were assembled and annotated for the differentially expressed genes (DEGs) analysis and novel transcripts identification. The result indicated that with the CaCO3 addition, the tricarboxylic cycle (TCA) cycle and glyoxylate pathway were up-regulated, and it also found that a non-ribosomal peptide synthetase (NRPS) like protein was highly expressed. The DEGs analysis showed a high expression level of malate dehydrogenase (MDHC) and phosphoenolpyruvate carboxykinase (PCKA) in the CaCO3 group, which indicated a cytosolic malate activity. We speculated that the malate should be transported to or synthesized in the cytoplasm, which was then polymerized to PMLA by the NRPS-like protein, accompanied by the up-regulated TCA cycle providing ATP for the polymerization. Depending on the analysis, we assumed that an NRPS-like protein, the TCA cycle, and the cytosolic malate together are contributing to the PMLA biosynthesis.

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Introduction: Pullulan is a biodegradable biopolymer made up of maltotriose subunits that are water-soluble. It has a direct glucan structure comprising α-1,4 and α-1,6 linkages in a 2:1 ratio, which gives it exceptional physical and structural properties, making it a suitable choice for application in biomedical applications. Despite its potential benefits, the price of production is expansive, and productivity is low, which are significant drawbacks. Therefore, the objective of this work is to identify the ideal carbon source for Aureobasidium melanogenum DSM 2404 to produce high pullulans. Methods: In shake flask studies, the effects of different carbon sources, such as maltose, glucose, sucrose, lactose, and xylose, on cell growth and pullulan generation by Aureobasidium melanogenum DSM 2404 were examined. Results: The highest pullulan production was obtained from sucrose (13.38 g L-1) compared to other types of carbon sources used in the fermentation medium. The maximal pullulan production rate of 0.112 [g-1 L-1 h-1] was obtained in sucrose culture. This suggests that sucrose provides favorable conditions for the microorganisms to produce pullulan at a faster rate compared to other sugars. Conclusion: Sucrose was found to be the most efficient carbon source for the synthesis of pullulan using Aureobasidium melanogenum DSM 2404 generating 13.38 g L-1 of pullulan. [ABSTRACT FROM AUTHOR]

Copyright of Xinjiang Agricultural Sciences is the property of Xinjiang Agricultural Sciences Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

L -piperazic acid (L -Piz) is one of components in many bioactive compounds synthesized by Streptomyces spp. Therefore, it is very important to develop a sustainable and eco-friendly bioprocess to produce L -Piz from renewable glucose. In this study, it was found that the engineered Aureobasidium melanogenum DFAK-ArgB6 strain overexpressing the ArgB gene encoding N-acetylglutamate kinase could produce 3.0 g/L L -Piz from glucose during the fed-batch fermentation. The yield was 55.6 mg L -Piz/g of glucose and the productivity was 24.8 mg/L/h. This was the highest amount of L -Piz produced by microorganisms so far. Finally, the partially purified L -Piz had purity of 86.4%. [Display omitted] • L -piperazic acid (L -Piz) is one of components in many bioactive compounds. • The DFAK-ArgB6 strain could produce the highest amount of L -Piz (3.0 g/L). • The partially purified L -Piz was obtained. [ABSTRACT FROM AUTHOR]

Poly (β-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries. In this study, electrolytic stimulation assisted PMLA production was developed. Firstly, it was found that the pentavalent nitrogen source (i.e., NO 3 −) was more suitable for PMLA production. Secondly, a usual single-chamber bioelectric-fermentation system (BES) cannot improve PMLA production, which can only promote cell growth. Then, a new single-chamber BES with an external circulation was developed, where the PMLA metabolism was further intensified. Finally, the integration of NO 3 − addition and electrolytic stimulation mode (c) showed a positive synergy on the PMLA production. Compared to the case without NO 3 − addition and electrolytic stimulation, the PMLA production was increased by 22.9 % using the integrated process. Moreover, compared to the case without the electrolytic stimulation mode (c), it was revealed that the different genes involved in 12 metabolic subsystems using the integrated process, where 31 and 177 genes were up-regulated and down-regulated, respectively. The up-regulated genes were mainly participated in melanin metabolic process, catalase activity, and oxidoreductase activity. Hence, the integration of electrolytic stimulation represents a novel approach to improve PMLA production. • A novel single-chamber BES with an external circulation was developed. • An oxidation state inorganic nitrogen source was more suitable for the PMLA production. • Electrolytic stimulation promotes PMLA production from A. melanogenum ipe-1. • The integration of NO 3 − addition and electrolytic stimulation showed positive synergy. [ABSTRACT FROM AUTHOR]

Marine fungi represent an important and sustainable resource, from which the search for novel biological substances for application in the pharmacy or food industry offers great potential. In our research, novel polysaccharide (AUM-1) was obtained from marine Aureobasidium melanogenum SCAU-266 were obtained and the molecular weight of AUM-1 was determined to be 8000 Da with 97.30% of glucose, 1.9% of mannose, and 0.08% galactose, owing to a potential backbone of α-D-Glcp-(1→2)-α-D-Manp-(1→4)-α-D-Glcp-(1→6)-(SO3−)-4-α-D-Glcp-(1→6)-1-β-D-Glcp-1→2)-α-D-Glcp-(1→6)-β-D-Glcp-1→6)-α-D-Glcp-1→4)-α-D-Glcp-6→1)-[α-D-Glcp-4]26→1)-α-D-Glcp and two side chains that consisted of α-D-Glcp-1 and α-D-Glcp-(1→6)-α-D-Glcp residues. The immunomodulatory effect of AUM-1 was identified. Then, the potential molecular mechanism by which AUM-1 may be connected to ferroptosis was indicated by metabonomics, and the expression of COX2, SLC7A11, GPX4, ACSL4, FTH1, and ROS were further verified. Thus, we first speculated that AUM-1 has a potential effect on the ferroptosis-related immunomodulatory property in RAW 264.7 cells by adjusting the expression of GPX4, regulated glutathione (oxidative), directly causing lipid peroxidation owing to the higher ROS level through the glutamate metabolism and TCA cycle. Thus, the ferroptosis related immunomodulatory effect of AUM-1 was obtained. [ABSTRACT FROM AUTHOR]

Background The great diversity of lifestyles and survival strategies observed in fungi is reflected in the many ways in which they reproduce and recombine. Although a complete absence of recombination is rare, it has been reported for some species, among them 2 extremotolerant black yeasts from Dothideomycetes: Hortaea werneckii and Aureobasidium melanogenum. Therefore, the presence of diploid strains in these species cannot be explained as the product of conventional sexual reproduction. Results Genome sequencing revealed that the ratio of diploid to haploid strains in both H. werneckii and A. melanogenum is about 2:1. Linkage disequilibrium between pairs of polymorphic loci and a high degree of concordance between the phylogenies of different genomic regions confirmed that both species are clonal. Heterozygosity of diploid strains is high, with several hybridizing genome pairs reaching the intergenomic distances typically seen between different fungal species. The origin of diploid strains collected worldwide can be traced to a handful of hybridization events that produced diploids, which were stable over long periods of time and distributed over large geographic areas. Conclusions Our results, based on the genomes of over 100 strains of 2 black yeasts, show that although they are clonal, they occasionally form stable and highly heterozygous diploid intraspecific hybrids. The mechanism of these apparently rare hybridization events, which are not followed by meiosis or haploidization, remains unknown. Both extremotolerant yeasts, H. werneckii and even more so A. melanogenum , a close relative of the intensely recombining and biotechnologically relevant Aureobasidium pullulans , provide an attractive model for studying the role of clonality and ploidy in extremotolerant fungi. [ABSTRACT FROM AUTHOR]

Abstract β-poly(l-malic acid) (PMLA) is a water-soluble biopolymer used in medicine, food, and other industries. However, the low level of PMLA biosynthesis in microorganisms limits its further application in the biotechnological industry. In this study, corn steep liquor (CSL), which processes high nutritional value and low-cost characteristics, was selected as a growth factor to increase the PMLA production in strain, Aureobasidium melanogenum, and its metabolomics change under the CSL addition was investigated. The results indicated that, with 3 g/L CSL, PMLA production, cell growth, and yield (Y p/x ) were increased by 32.76%, 41.82%, and 47.43%, respectively. The intracellular metabolites of A. melanogenum, such as amino acids, organic acids, and key intermediates in the TCA cycle, increased after the addition of CSL, and the enrichment analysis showed that tyrosine may play a major role in the PMLA biosynthesis. The results presented in this study demonstrated that the addition of CSL would be an efficient approach to improve PMLA production.

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.

Aureobasidium melanogenum P16, the high pullulan producer, had only one GATA type transcriptional activator AreA and one GATA type transcriptional repressor AreB. It was found that 2.4 g/L of (NH 4) 2 SO 4 had obvious nitrogen repression on pullulan biosynthesis by A. melanogenum P16. Removal of the AreB gene could make the disruptant DA6 produce 34.8 g/L pullulan while the P16 strain only produced 28.8 g/L pullulan at the efficient nitrogen condition. Further both removal of the native AreA gene and overexpression of the mutated AreA S628-S678 gene with non-phosphorylatable residues could render the transformant DEA12 to produce 39.8 g/L pullulan. The transcriptional levels of most of the genes related to pullulan biosynthesis in the transformant DEA12 were greatly enhanced. The mutated AreA S628-S678 was localized in the nuclei of the transformant DEA12 while the native AreA was distributed in the cytoplasm in A. melanogenum P16. This meant that nitrogen repression on pullulan biosynthesis in the transformant DEA12 was indeed significantly relieved. This was the first time to report that the GATA type transcriptional factors of nitrogen catabolite repression system could regulate pullulan biosynthesis in Aureobasidium spp. • The GATA type transcriptional activator AreA and the transcriptional repressor AreB were removed. • The mutated AreA gene with nonphosphorylatable residues was overexpressed. • The obtained derepressed mutant could produce 39.8 g/L pullulan. • The mutated AreA was only localized in the nuclei of the derepressed mutant. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • Many melanin-synthesizing genes are linked together as the PKS gene clusters. • Most of the PKS gene clusters are regulated by Cmr1. • Evolution of different genes in the clusters is closely related to adaptation of the melanin-producing fungi. [ABSTRACT FROM AUTHOR]

In this study, an NSDD gene, which encoded a GATA-type transcription factor involved in the regulation and biosynthesis of melanin, pullulan, and polymalate (PMA) in Aureobasidium melanogenum , was characterized. After the NSDD gene was completely removed, melanin production by the Δnsd mutants was enhanced, while pullulan and polymalate production was significantly reduced. Transcription levels of the genes involved in melanin biosynthesis were up-regulated while expression levels of the genes responsible for pullulan and PMA biosynthesis were down-regulated in the Δnsdd mutants. In contrast, the complementation of the NSDD gene in the Δnsdd mutants made the overexpressing mutants restore melanin production and transcription levels of the genes responsible for melanin biosynthesis. Inversely, the complementation strains, compared to the wild type strains, showed enhanced pullulan and PMA yields. These results demonstrated that the NsdD was not only a negative regulator for melanin biosynthesis, but also a key positive regulator for pullulan and PMA biosynthesis in A. melanogenum. It was proposed how the same transcriptional factor could play a negative role in melanin biosynthesis and a positive role in pullulan and PMA biosynthesis. This study provided novel insights into the regulatory mechanisms of multiple A. melanogenum metabolites and the possibility for improving its yields of some industrial products through genetic approaches. • The NSDD genes from different strains of A. melanogenum were characterized. • NsdD is a positive regulator for pullulan and polymarate biosynthesis. • However, NsdD is a negative regulator for melanin biosynthesis. • The negative regulation mode by NsdD was proposed. [ABSTRACT FROM AUTHOR]

Selenium (Se) plays a critical role in diverse biological processes and is widely used across manufacturing industries. However, the contamination of Se oxyanions also poses a major public health concern. Microbial transformation is a promising approach to detoxify Se oxyanions and produce elemental selenium nanoparticles (SeNPs) with versatile industrial potential. Yeast-like fungi are an important group of environmental microorganisms, but their mechanisms for Se oxyanions reduction remain unknown. In this study, we found that Aureobasidium melanogenum I15 can reduce 1.0 mM selenite by over 90% within 48 h and efficiently form intracellular or extracellular spherical SeNPs. Metabolomic and proteomic analyses disclosed that A. melanogenum I15 evolves a complicated selenite reduction mechanism involving multiple metabolic pathways, including the glutathione/glutathione reductase pathway, the thioredoxin/thioredoxin reductase pathway, the siderophore-mediated pathway, and multiple oxidoreductase-mediated pathways. This study provides the first report on the mechanism of selenite reduction and SeNPs biogenesis in yeast-like fungi and paves an alternative avenue for the bioremediation of selenite contamination and the production of functional organic selenium compounds. [Display omitted] • melanogenum I15 can reduce over 90% of 1 mM selenite within 48 h. • Selenite reduction occurs both intracellularly and extracellularly in I15. • Selenite reduction occurs through multiple oxidoreductase-mediated pathways in I15. • Selenite stress alters the cell morphology and the cell membrane structure of I15. • Glutathione and siderophores were actively involved in the selenite reduction of I15. [ABSTRACT FROM AUTHOR]

Marine fungi represent an important and sustainable resource, from which the search for novel biological substances for application in the pharmacy or food industry offers great potential. In our research, novel polysaccharide (AUM-1) was obtained from marine Aureobasidium melanogenum SCAU-266 were obtained and the molecular weight of AUM-1 was determined to be 8000 Da with 97.30% of glucose, 1.9% of mannose, and 0.08% galactose, owing to a potential backbone of α-D-Glcp-(1→2)-α-D-Manp-(1→4)-α-D-Glcp-(1→6)-(SO3−)-4-α-D-Glcp-(1→6)-1-β-D-Glcp-1→2)-α-D-Glcp-(1→6)-β-D-Glcp-1→6)-α-D-Glcp-1→4)-α-D-Glcp-6→1)-[α-D-Glcp-4]26→1)-α-D-Glcp and two side chains that consisted of α-D-Glcp-1 and α-D-Glcp-(1→6)-α-D-Glcp residues. The immunomodulatory effect of AUM-1 was identified. Then, the potential molecular mechanism by which AUM-1 may be connected to ferroptosis was indicated by metabonomics, and the expression of COX2, SLC7A11, GPX4, ACSL4, FTH1, and ROS were further verified. Thus, we first speculated that AUM-1 has a potential effect on the ferroptosis-related immunomodulatory property in RAW 264.7 cells by adjusting the expression of GPX4, regulated glutathione (oxidative), directly causing lipid peroxidation owing to the higher ROS level through the glutamate metabolism and TCA cycle. Thus, the ferroptosis related immunomodulatory effect of AUM-1 was obtained.

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.

Abstract 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.

Summary: 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. [ABSTRACT FROM AUTHOR]

A novel yeast strain Aureobasidium melanogenum 13-2 isolated from the Taklimakan desert was found to be able to produce a high level of extracellular polysaccharide (EPS). Under the optimal conditions, the yeast strain could yield 73.25 ± 2.3 g/L of EPS within 5 days at a flask level. During a 10-liter fermentation, the yeast strain could produce 78.05 ± 3.5 g/L of EPS within 120 h. The FT-IR spectra of the standard pullulan from Sigma and the purified EPS produced by A. melanogenum 13-2 were almost identical and the purified EPS could be actively hydrolyzed by a pullulanase, demonstrating that the purified EPS was pullulan. The molecular weight (Mw) of the purified pullulan was estimated to be 7.703 × 105 g/moL. Disruption of a pullulan synthase gene (PUL1) made a mutant DAG27 lose the ability to synthesize any pullulan. The mutant DAG27 was more sensitive to radiation of UV light, high NaCl concentration, heat treatment, strong oxidation of H 2 O 2 and desiccation than its wild type strain 13-2, suggesting that the produced pullulan could play an important role in resistance of the yeast cells to various stresses. This was the first time to show that the yeast strain obtained from the desert could produce such high level pullulan and the produced pullulan had an obviously protective effect on its producer. • A novel yeast strain 13-2 produced 78.05 ± 3.5 g/L of pullulan. • Disruption of a pullulan synthase gene (PUL1) made a mutant DAG27 synthesize no pullulan. • The produced pullulan played an important role in resistance of the yeast cells to various stresses. [ABSTRACT FROM AUTHOR]

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384 × 106 Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30 °C and 180 rpm: carbon source, 50 g/L maltose; initial pH 7; and 8 g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3 L. The fermentation broth contained 303 g/L maltose, which produced 122.34 g/L pullulan with an average productivity of 1.0195 g/L/h and 82.32 g/L dry biomass within 120 h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251 g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production. [ABSTRACT FROM AUTHOR]

Overexpression of the optimized Vitreoscilla hemoglobin (VHb) gene and the native flavohemoglobin (FHb) gene in Aureobasidium melanogenum P16 rendered a V6 strain and a F44 strain to overproduce pullulan compared to that produced by their wild type strain P16. The capacity to bind CO and oxygen in the V6 strain and the F44 strain was also obviously enhanced. At the same time, the transcriptional levels of the relevant genes were also increased in the V6 strain and the F44 strain and the fused vgbop + the gene encoding GFP and FHb gene + the gene encoding GFP were also actively expressed. During a 10-liter fermentation, the P16 strain produced only 72.0 ± 1.0 g/L pullulan, the yield was 0.77 g/g of sucrose, the productivity was 0.5 ± 0.01 g/L/h and only 79.4% of the total sugar was used. In contrast, the strain V6 yielded 102.3 ± 1.8 g/L of pullulan, the yield was 0.89 g/g of sucrose, the productivity was 0.7 ± 0.01 g/L/h and 96.0% of total sugar was used while 101.4 ± 2.9 g/L of pullulan was accumulated in the culture of the strain F44, the yield was 0.88 g/g of sucrose, the productivity was 0.7 ± 0.02 g/L/h and 96.4% of total sugar was utilized. These data strongly demonstrated that the concentration of pullulan, yield, productivity and sugar utilization were greatly enhanced by overexpression of the VHb and FHb. But their cell growth was almost the similar. • The optimized vgbop gene and the native FHb gene were overexpressed. • Pullulan and the capacity to bind CO and oxygen were greatly enhanced. • The pullulan concentrations, productivity and sugar utilization were also greatly enhanced. [ABSTRACT FROM AUTHOR]

Aureobasidium melanogenum 11–1 was found to be able to produce over 281.7 ± 7.1 U/mL of β-fructofuranosidase activity. The protein deduced from the cloned β - fructofuranosidase1 gene had the conserved motif A (IGDP), motif D (RDP) and motif E (ET) and 11 N-glycosylation sites, indicating it was a β-fructofuranosidase with the high-level fructooligosaccharide (FOS) biosynthesis. Overexpression of the β - fructofuranosidase1 gene in the yeast strain 11–1 made a tranformant 33 produce 557.7 U/mL of β-fructofuranosidase activity. The molecular weight of the β-fructofuranosidase1 in which all the carbohydrates were removed by the Endo-H was 82.4 kDa. Within 7 h of the transfructosylation reaction, the yield of FOS was 0.66 g of FOS/g of sucrose and percentages of GF2, GF3 and GF4 were 79.5%, 18.9% and 1.6%. This demonstrated that the β-fructofuranosidase1 and the transformant 33 had highly potential applications in biotechnology for FOS production. • The gene encoding β-fructofuranosidase1 with the high-level fructooligosaccharide (FOS) biosynthesis was overexpressed. • 557.7 U/mL of β-fructofuranosidase was produced within 60 h. • The yield of FOS was 0.66 g of FOS/g of sucrose and most of the FOSs were GF2 (79.5%) and GF3 (18.9%). [ABSTRACT FROM AUTHOR]

Poly(β-L-malic acid) (PMA) is a promising polyester formed from L-malate in microbial cells. Malate biosynthesis is crucial for PMA production. Previous studies have shown that the non-oxidative pathway or oxidative pathway (TCA cycle) is the main biosynthetic pathway of malate in most of PMA-producing strains, while the glyoxylate cycle is only a supplementary pathway. In this study, we investigated the effect of exogenous metabolic intermediates and inhibitors of the malate biosynthetic pathway on PMA production by Aureobasidium melanogenum GXZ-6. The results showed that PMA production was stimulated by maleic acid (a fumarase inhibitor) and sodium malonate (a succinate dehydrogenase inhibitor) but inhibited by succinic acid and fumaric acid. This indicated that the TCA cycle might not be the only biosynthetic pathway of malate. In addition, the PMA titer increased by 18.1% upon the addition of glyoxylic acid after 72 h of fermentation, but the PMA titer decreased by 7.5% when itaconic acid (an isocitrate lyase inhibitor) was added, which indicated that malate for PMA production was synthesized significantly via the glyoxylate cycle rather than the TCA cycle. Furthermore, in vitro enzyme activities of the TCA and glyoxylate cycles suggested that the glyoxylate cycle significantly contributed to the PMA production, which is contradictory to what has been reported previously in other PMA-producing A. pullulans. [ABSTRACT FROM AUTHOR]

Abstract It has been thought that when different strains of Aureobasidium spp. were grown in sucrose, the produced fructooligosaccharides (FOSs) by β- d -fructofuranosidase were beneficial for their cell growth and pullulan biosynthesis. However, it is still unknown about how β- d -fructofuranosidases activity and synthesized FOSs influence on pullulan biosynthesis. It was found that the genomic DNA of Aureobasidium melanogenum P16, a high pullulan producing yeast, contained three genes encoding β- d -fructofuranosidase1, β- d -fructofuranosidase2 and β- d -fructofuranosidase3. The FTR1 factor, a transcriptional activator, activated expression of the three β- d -fructofuranosidase genes and invertase gene. Disruption of the FTR1 gene rendered a disruptant DF3 to produce less FOSs (12.1 ± 0.4 g/L), less β- d -fructofuranosidase activity (1.1 ± 0.2 U/mL), lower Mw (3.8 × 105) of the pullulan and more pullulan titer (77.0 ± 2.6 g/L) than the yeast strain P16. Similarly, removal of both the two genes encoding β- d -fructofuranosidase1 and β- d -fructofuranosidase3 resulted in a double mutant DF4–7 producing 77.5 ± 3.1 g/L pullulan with Mw of 3.4 × 105, 0.2 ± 0.0 U/mL of β- d -fructofuranosidase activity and the trace amount of FOSs while its wild type strain P16 yielded 65.7 ± 3.5 g/L pullulan with Mw of 4.4 × 105, 6.8 ± 0.0 U/mL of β- d -fructofuranosidase activity and 6.2 ± 0.5 g/L of FOSs. These confirmed that high β- d -fructofuranosidase activity, the presence of high level of FOSs negatively influenced pullulan biosynthesis, but positively increased Mw of the produced pullulan. However, the β- d -fructofuranosidase2 had no such function. Furthermore, complementation of the FTR1 gene, β- d -fructofuranosidase1 gene and β- d -fructofuranosidase3 gene enabled the corresponding transformants to restore β- d -fructofuranosidase activity, FOSs and pullulan biosynthesis and Mw of the pullulan. Highlights • High β- d -fructofuranosidase activity and the presence of high-level FOSs negatively influenced pullulan biosynthesis • The presence of high-level FOSs positively increased Mw (4.4 × 105) of the produced pullulan • β- d -fructofuranosidase and invertase were induced by a transcriptional activator. [ABSTRACT FROM AUTHOR]

Highlights • Three fermentation strategies for PMA production from malt syrup were studied. • High PMA titer 124.07 g/L was obtained by fed-batch fermentation. • PMA yield 1.10 g/g and productivity 0.70 g/L·h in repeated-batch fermentation. • PMA production cost is $ 1.716/kg by fed-batch fermentation at 2000 MT plant. • This provides an economical strategy for large-scale PMA production. Abstract Malt syrup, as a low-cost substrate without any pretreatment, was proved to be able to replace maltose for ploymalic acid (PMA) production by Aureobasidium melanogenum GXZ-6. The PMA titer of 55.53 ± 1.72 g/L was obtained by batch fermentation in a 10-L fermentor with addition of malate, citrate and sodium malonate. Then, a higher PMA titer of 124.07 ± 2.28 g/L was obtained in fed-batch fermentation, which increased by 123.43% than that from batch fermentation. Moreover, repeated-batch fermentation with three batches gave a PMA titer of 64.06 g/L on average with a higher yield of 0.81 g/g and productivity of 0.56 g/L·h. Fermentation process and economics analysis were performed by SuperPro Designer for a 2000 metric tons plant. Results showed that PMA production cost was as low as $ 1.716/kg by fed-batch fermentation, which provides an economical strategy for large-scale PMA production. [ABSTRACT FROM AUTHOR]

Aureobasidium melanogenum strain 11-1 with a high laccase activity was isolated from a mangrove ecosystem. Under the optimal conditions, the 11-1 strain yielded the highest laccase activity up to 3120.0 ± 170 mU/ml (1.2 U/mg protein) within 5 days. A laccase gene (LAC1) of the yeast strain 11-1 contained two introns and encoded a protein with 570 amino acids and four conserved copper-binding domains typical of the fungal laccase. Expression of the LAC1 gene in the yeast strain 11-1 made a recombinant yeast strain produce the laccase activity of 6005 ± 140 mU/ml. The molecular weight of the recombinant laccase after removing the sugar was about 62.5 kDa. The optimal temperature and pH of the recombinant laccase were 40 °C and 3.2, respectively, and it was stable at a temperature less than 25 °C. The laccase was inhibited in the presence of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), and DL-dithiothreitol (DTT). The Km and Vmax values of the laccase for 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was 6.3 × 10−2 mM and 177.4 M/min, respectively. Many synthetic dyes were greatly decolored by the laccase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pullulan, which is a microbial exopolysaccharide, has found widespread applications in foods, biomedicines, and cosmetics. Despite its versatility, most wild-type strains tend to yield low levels of pullulan production, and their mutants present genetic instability, achieving a limited increase in pullulan production. Therefore, mining new wild strains with robust pullulan-producing abilities remains an urgent concern. In this study, we found a novel strain, namely, Aureobasidium melanogenum ZH27, that had a remarkable pullulan-producing capacity and optimized its cultivation conditions using the one-factor-at-a-time method. To elucidate the reasons that drove the hyper-production of pullulan, we scrutinized changes in cell morphology and gene expressions. The results reveal that strain ZH27 achieved 115.4 ± 1.82 g/L pullulan with a productivity of 0.87 g/L/h during batch fermentation within 132 h under the optimized condition (OC). This pullulan titer increased by 105% compared with the initial condition (IC). Intriguingly, under the OC, swollen cells featuring 1-2 large vacuoles predominated during a rapid pullulan accumulation, while these swollen cells with one large vacuole and several smaller ones were prevalent under the IC. Moreover, the expressions of genes associated with pullulan accumulation and by-product synthesis were almost all upregulated. These findings suggest that swollen cells and large vacuoles may play pivotal roles in the high level of pullulan production, and the accumulation of by-products also potentially contributes to pullulan synthesis. This study provides a novel and promising candidate for industrial pullulan production.

In this study, in order to directly and efficiently convert inulin into a single-cell oil (SCO), an INU1 gene encoding inulinase fromKluyveromyces marxianus was integrated into the genomic DNA and actively expressed in an SCO producer Aureobasidiummelanogenum P10. The transformant API41 obtained produced 28.5 U/mL of inulinase and its wild-type strain P10 yielded only 8.62 U/mL. Most (97.5%) of the inulinase produced by the transformant API41 was secreted into the culture. During a 10-L fermentation, 66.2% (w/w) lipid in the yeast cells of the transformant API41 and 14.38 g/L of cell dry weight were attained from inulin of 80.0 g/L within 120 h, high inulinase activity (23.7 U/mL) was also produced within 72 h, and the added inulin was actively hydrolyzed. This confirmed that the genetically engineered yeast of A. melanogenum P10 is suitable for direct production of lipids from inulin. The lipids produced could be used as feedstocks for biodiesel production. [ABSTRACT FROM AUTHOR]

A very high pullulan producing yeast-like fungus, Aureobasidium melanogenum TN1-2 isolated from a natural honey was found to be able to produce 97.0 g/L of pullulan from 140.0 g/L sucrose at a flask level while it could yield 114.0 g/L of pullulan within 134 h during a 10-liter fermentation, the yield was 0.81 g/g and the productivity was 0.86 g/L/h. The high ability to biosynthesize pullulan by this yeast-like fungal strain TN1-2 was related to high glucosyltransferase activity, high phosphofructo-2-kinase activity, high content of its cellular glycerol and low glucose repressor. The Mw of the produced pullulan was 1.42 × 10 5  g/mol. The low Mw may be due to the high α-amylase, glucoamylase and isopullulanase activities. The intracellular level of trehalose had no influence on high pullulan production by the yeast-like fungal strain TN1-2. [ABSTRACT FROM AUTHOR]

Melanin plays an important role in the stress adaptation of Aureobasidium melanogenum XJ5-1 isolated from the Taklimakan desert. A trehalose-6-phosphate synthase gene (TPS1 gene) was cloned from K5, characterized, and then deleted to determine the role of trehalose in the stress adaptation of the albino mutant K5. No stress response element and heat shock element were found in the promoter of the TPS1 gene. Deletion of the TPS1 gene in the albino mutant rendered a strain DT43 unable to synthesize any trehalose, but DT43 still could grow in glucose, suggesting that its hexokinase was insensitive to inhibition by trehalose-6-phosphate. Overexpression of the TPS1 gene enhanced trehalose biosynthesis in strain ET6. DT43 could not grow at 33 °C, whereas K5, ET6, and XJ5-1 could grow well at this temperature. Compared with K5 and ET6, DT43 was highly sensitive to heat shock treatment, high oxidation, and high desiccation, but all the three strains demonstrated the same sensitivity to UV light and high NaCl concentration. Therefore, trehalose played an important role in the adaptation of K5 to heat shock treatment, high oxidation, and high desiccation. [ABSTRACT FROM AUTHOR]

Tannins biodegradation by a microorganism is one of the most efficient ways to produce bioproducts of high value. However, the mechanism of tannins biodegradation by yeast has been little explored. In this study, Aureobasidium melanogenum T9 isolated from red wine starter showed the ability for tannins degradation and had its highest biomass when the initial tannic acid concentration was 20 g/L. Furthermore, the genes involved in the tannin degradation process were analyzed. Genes tan A, tan B and tan C encoding three different tannases respectively were identified in the A. melanogenum T9. Among these genes, tan A and tan B can be induced by tannin acid simultaneously at both gene transcription and protein expression levels. Our assay result showed that the deletion of tanA and tanB resulted in tannase activity decline with 51.3 ± 4.1 and 64.1 ± 1.9 U/mL, respectively, which is much lower than that of A. melanogenum T9 with 91.3 ± 5.8 U/mL. In addition, another gene coding gallic acid decarboxylase (gad) was knocked out to better clarify its function. Mutant Δgad completely lost gallic acid decarboxylase activity and no pyrogallic acid was seen during the entire cultivation process, confirming that there was a sole gene encoding decarboxylase in the A. melanogenum T9. These results demonstrated that tanA, tanB and gad were crucial for tannin degradation and provided new insights for the mechanism of tannins biodegradation by yeast. This finding showed that A. melanogenum has potential in the production of tannase and metabolites, such as gall acid and pyrogallol.

Aureobasidium melanogenum P16 is a high pullulan-producing yeast. However, glucose repression on its pullulan biosynthesis must be relieved. After the gene encoding a glucose repressor was cloned, characterized and analyzed, it was found that the repressor belonged to one member of the CreA in filamentous fungi, not to one member of the Mig1 in yeasts. After the CREA gene was fully removed from the yeast strain P16, the glucose repression in the disruptant DG41 was relieved. At the same time, the pullulan production by the disruptant DG41 was enhanced compared to that by its wild-type strain P16, and the transcriptional levels of the gene encoding a glucosyltransferase, three genes encoding glucose transporters, the gene encoding a 6-P-glucose kinase and the genes encoding α-amylase, glucoamylase and pullulanase in the disruptant DG41 were also promoted. However, the transcriptional levels of the genes encoding the CreA and another two glucose transporters were greatly reduced. During the 10-liter fermentation, the disruptant DG41 produced 64.93 ± 1.33 g/l pullulan from 120 g/l of glucose, while its wild-type strain P16 produced only 52.0 ± 1.95 g/l pullulan within 132 h. After the CREA gene was complemented in the disruptant D373, the pullulan production by the transformant BC4 was greatly reduced compared to that by its wild-type strain P16, and the transcriptional levels of the many genes in the transformant BC4 were also decreased. All the results confirmed that the CreA played an important role in the regulation of pullulan biosynthesis in A. melanogenum P16, and that glucose derepression on pullulan biosynthesis could improve pullulan production from glucose. This study opened the possibility for improving the industrial production of this exopolysaccharide by genetic engineering. [ABSTRACT FROM AUTHOR]

Aureobasidium melanogenum HN6.2 was found to able to produce an esterase of 208.1 ± 2.7 units per ml within 72 h. A molecular weight of the purified esterase was 60.2 kDa and its PI was 4.86. The optimal pH and temperature of the purified esterase were 8.0 and 40 °C, respectively. The purified esterase was stable at the temperature less than 40 °C and in the pH range of 7.5–8.0. The esterase activity was greatly inhibited in the presence of Zn 2+ , Hg 2+ , Fe 2+ , Ni 2+ and SDS. K m and V max values of the enzyme for ρ-nitrophenyl butyrate were 68.6 μM and 251.4 μM per min, respectively. Mass analysis showed that it belonged to a carboxylesterase. After an esterase gene ( Car-Est ) cloned from the genomic DNA of the marine yeast strain HN6.2 was disrupted, the disruptant Y44 obtained exhibited a significant decrease in esterase activity. Meanwhile, a transcriptional level of the Car-Est gene of the disruptant Y44 was only 5.18% that of the Car-Est gene of its wild type strain HN6.2, confirming that the cloned esterase gene was indeed closely related to the esterase activity of the yeast HN6.2 strain. [ABSTRACT FROM AUTHOR]

Aureobasidium melanogenum is a saprophytic, dematiaceous, yeast-like fungus rarely implicated in human infections. Here, we report the first case of A. melanogenum fungemia in a 30-week-old preterm, very low birth weight neonate born to a primigravida with history of gestational diabetes, pregnancy induced hypertension and oligohydramnios. The baby developed respiratory distress, hypotension, bradycardia, coagulopathy and septic shock shortly after birth, and eventually succumbed to multiple organ dysfunction syndrome on day 9 of life. Paired blood culture showed growth of a dematiaceous yeast-like fungus which was identified as A. melanogenum by rDNA internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing of the isolate showed high minimum inhibitory concentration of fluconazole (32 µg/mL), indicating resistance. Diagnosis of A. melanogenum fungemia is difficult as it is easily confused with Candida species in Gram stained smears and similar colony morphology during the initial stages of growth. Also, the conventional diagnostic methods, such as VITEK 2 and MALDI-TOF MS are unreliable for identification of this pathogen. Accurate identification using molecular techniques is crucial for making treatment decisions as A. melanogenum shows substantial antifungal resistance. Clinicians should be aware that yeast-like cells in blood culture are not only indicative of Candida species, but also rare pathogens like A. melanogenum and should exercise caution while starting fluconazole therapy. At present, there are no established susceptibility breakpoints for Aureobasidium spp. Further studies are needed to determine the optimal treatment for such infections. [ABSTRACT FROM AUTHOR]

cDNA ofAureobasidium melanogenumlipase 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. TheA. melanogenumlipase gene was successfully expressed inPichia 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 usingp-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. Lipase activity ofAureobasidium melanogenumlipase expressed inPichia pastorisusing inducible and constitutive expression system. [ABSTRACT FROM PUBLISHER]

Aureobasidium pullulans is a ubiquitous black yeast-like fungus belonging to order Dothideales. It was regarded as a contaminant, but is now considered a pathogen causing a wide range of human infections. We report a case of superficial phaeohyphomycosis in an immunocompetent patient with clinical presentations mimicking tinea nigra. On microscopic examination of lesion scales, multiple thick-walled, pigmented oval spores with septa were noted. A fungus with black mucoid colonies was repeatedly isolated from the lesions during the treatment course. This fungus was identified as A. melanogenum on the basis of morphological characteristics and subsequently confirmed by sequencing internal transcribed spacers of ribosomal DNA. The clinical presentations and microscopic findings of lesion scales were considerably similar to those of tinea nigra. However, fungal culturing proved that the causative pathogen was A. melanogenum rather than Hortaea werneckii. The patient might have acquired this infection during gardening activities. We also reviewed reported cases of cutaneous A. pullulans infection. [ABSTRACT FROM AUTHOR]