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.

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.

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.

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.

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., NO3-) 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 NO3- addition and electrolytic stimulation mode (c) showed a positive synergy on the PMLA production. Compared to the case without NO3- 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.

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.

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.
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.
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.

In this study, it was found that reducing consumption of acetyl-CoA in mitochondria, peroxisome and lipid biosynthesis could not obviously enhance liamocin biosynthesis by engineered strains of Aureobasidium melanogenm 9-1, but decreased cell growth of the mutants. On the contrary, expression of heterologous PTA gene for phosphotransacetylase in PK pathway and native ALD gene for acetaldehyde dehydrogenase and ACS gene encoding acetyl-CoA synthetase in the PDH bypass pathway reduced liamocin biosynthesis. However, expression the PK gene for phosphoketolase, the PDC gene encoding pyruvate decarboxylase and VHb gene coding for Vitreoscilla hemoglobin (VHb) in the glucose derepression mutants could greatly enhance liamocin production. The resulting strain V33 could produce 55.38 g/L of liamocin and 25.10 g/L of cell dry weight from 117.27 g/L of glucose within 168 h of 10-liter fermentation, leading to the yield of 0.47 g/g of glucose, the productivity of 0.33 g/L/h and rate of glucose utilization of 0.70 ± 0.01 g/L/h. This was a new and efficient strategy for overproduction of liamocin by A. melanogenm.

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.

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.