We here explore the potential of the fungal genus Aureobasidium as a prototype for a microbial chassis for industrial biotechnology in the context of a developing circular bioeconomy. The study emphasizes the physiological advantages of Aureobasidium, including its polyextremotolerance, broad substrate spectrum, and diverse product range, making it a promising candidate for cost-effective and sustainable industrial processes. In the second part, recent advances in genetic tool development, as well as approaches for up-scaled fermentation, are described. This review adds to the growing body of scientific literature on this remarkable fungus and reveals its potential for future use in the biotechnological industry.

Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/μg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism.

Aureobasidium pullulans is a ubiquitous fungus with a wide variety of morphologies and growth modes including \"typical\" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of A. pullulans promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for A. pullulans, which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.

Different preventive strategies are needed to minimize the intake risks of mycotoxins, including zearalenone (ZEN). The aim of this study was to determine the ZEN adsorption ability of an autolyzed biomass preparation of polymorphic yeast Aureobasidium pullulans A.p.-3. The evaluation of the antitoxic properties of the preparation was also performed in relation to Saccharomyces cerevisiae yeast (ATCC 2366, ATCC 7090 and ATCC 9763) used as a model cell exposed to a toxic ZEN dose. The preparation at a dose of 5 mg/mL showed the adsorption of ZEN present in model systems at concentrations between 1 μg/mL to 100 μg/mL. The highest degree of adsorption was established for ZEN concentrations of 1 μg/mL and 5 μg/mL, becoming limited at higher doses of the toxin. Based on the Langmuir model of adsorption isotherms, the predicted maximum ZEN adsorption was approx. 190 µg/mL, regardless of pH. The growth of three strains of S. cerevisiae yeast cells in the medium with ZEN at concentrations within the range of 1.56 μg/mL-100 μg/mL was analyzed to determine the minimum inhibitory concentration. The growth of all tested strains was especially limited by high doses of ZEN, i.e., 50 and 100 μg/mL. The protective effect of the tested preparation was noted in relation to yeast cells exposed to toxic 100 μg/mL ZEN doses. The highest yeast cell growth (app. 36% percentage) was noted for a S. cerevisiae ATCC 9763 strain compared to the medium with ZEN but without preparation. More detailed tests determining the antitoxic mechanisms of the A. pullulans preparation are planned in the future, including cell culture bioassays and animal digestive tract models.

Aureobasidium is omnipresent and can be isolated from air, water bodies, soil, wood, and other plant materials, as well as inorganic materials such as rocks and marble. A total of 32 species of this fungal genus have been identified at the level of DNA, of which Aureobasidium pullulans is best known. Aureobasidium is of interest for a sustainable economy because it can be used to produce a wide variety of compounds, including enzymes, polysaccharides, and biosurfactants. Moreover, it can be used to promote plant growth and protect wood and crops. To this end, Aureobasidium cells adhere to wood or plants by producing extracellular polysaccharides, thereby forming a biofilm. This biofilm provides a sustainable alternative to petrol-based coatings and toxic chemicals. This and the fact that Aureobasidium biofilms have the potential of self-repair make them a potential engineered living material avant la lettre. KEY POINTS: •Aureobasidium produces products of interest to the industry •Aureobasidium can stimulate plant growth and protect crops •Biofinish of A. pullulans is a sustainable alternative to petrol-based coatings •Aureobasidium biofilms have the potential to function as engineered living materials.

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.

Black yeasts have been isolated from acidic, low water activity, and thermally processed foods as well as from surfaces in food manufacturing plants. The genomic basis for their relative tolerance to food-relevant environmental stresses has not been well defined. In this study, we performed whole genome sequencing (WGS) on seven black yeast strains including Aureobasidium (n=5) and Exophiala (n=2) which were isolated from food or food production environments. These strains were previously characterized for their tolerance to heat, hyperosmotic pressure, high pressure processing, hypochlorite sanitizers, and ultraviolet light. Based on the WGS data, three of the strains previously identified as A. pullulans were reassigned as A. melanogenum. Both haploid and diploid A. melanogenum strains were identified in this collection. Single-locus phylogenies based on beta tubulin, RNA polymerase II, or translation elongation factor protein sequences were compared to the phylogeny produced through SNP analysis, revealing that duplication of the fungal genome in diploid strains complicates the use of single-locus phylogenetics. There was not a strong association between phylogeny and either environmental source or stress tolerance phenotype, nor were trends in the copy numbers of stress-related genes associated with extremotolerance within this collection. While there were obvious differences between the genera, the heterogenous distribution of stress tolerance phenotypes and genotypes suggests that food-relevant black yeasts may be ubiquitous rather than specialists associated with particular ecological niches. However, further evaluation of additional strains and the potential impact of gene sequence modification is necessary to confirm these findings.

Fungal keratitis is a time-sensitive ocular infection that often requires a high index of suspicion followed by intensive medical/surgical interventions to achieve a successful clinical outcome. COVID-19 pandemic-related restrictions, necessitated the modification of conventional protocols and guidelines associated with the treatment of keratomycosis. We report four cases of atypical fungal keratitis with poorly differentiated clinical characteristics. The challenges faced during their management were (1) the dilemma of clinically differentiating fungal (Scedosporium and Purpureocillium lilacinum) and bacterial keratitis; (2) treatment of Scedosporium and Trichosporon keratitis with natamycin monotherapy; (3) mixed infection of Candida albicans and Aureobasidium pullulans and continuing medications before rescraping the corneal ulcer against the recommended treatment guidelines; (4) phenotypic identification and differentiation among morphologically resembling fungi; and (5) decision making arising out of disparities between KOH and fungal culture results. Three patients responded well to conservative treatments. The fourth patient underwent therapeutic keratoplasty but was lost to follow-up due to travel-related pandemic restrictions. This case series seeks to broaden the clinician\'s knowledge of rare and emerging moulds as presumptive aetiologies of keratomycosis. It also intends to emphasize the significance of early microbiological investigations, (direct microscopy and culture), in resource-limited settings, for initiating empirical treatment for a better visual prognosis.

Fungicide applications in agriculture and medicine can promote the evolution of resistant, pathogenic fungi, which is a growing problem for disease management in both settings. Nonpathogenic mycobiota are also exposed to fungicides, may become tolerant, and could turn into agricultural or medical problems, for example, due to climate change or in immunocompromised individuals. However, quantitative data about fungicide sensitivity of environmental fungi is mostly lacking. Aureobasidium species are widely distributed and frequently isolated yeast-like fungi. One species, A. pullulans, is used as a biocontrol agent, but is also encountered in clinical samples, regularly. Here, we compared 16 clinical and 30 agricultural Aureobasidium isolates based on whole-genome data and by sensitivity testing with the 3 fungicides captan, cyprodinil, and difenoconazole. Our phylogenetic analyses determined that 7 of the 16 clinical isolates did not belong to the species A. pullulans. These isolates clustered with other Aureobasidium species, including A. melanogenum, a recently separated species that expresses virulence traits that are mostly lacking in A. pullulans. Interestingly, the clinical Aureobasidium isolates were significantly more fungicide sensitive than many isolates from agricultural samples, which implies selection for fungicide tolerance of non-target fungi in agricultural ecosystems. IMPORTANCE Environmental microbiota are regularly found in clinical samples and can cause disease, in particular, in immunocompromised individuals. Organisms of the genus Aureobasidium belonging to this group are highly abundant, and some species are even described as pathogens. Many A. pullulans isolates from agricultural samples are tolerant to different fungicides, and it seems inevitable that such strains will eventually appear in the clinics. Selection for fungicide tolerance would be particularly worrisome for species A. melanogenum, which is also found in the environment and exhibits virulence traits. Based on our observation and the strains tested here, clinical Aureobasidium isolates are still fungicide sensitive. We, therefore, suggest monitoring fungicide sensitivity in species, such as A. pullulans and A. melanogenum, and to consider the development of fungicide tolerance in the evaluation process of fungicides.

Polygalacturonase (PGase) production by Aureobasidium pullulans P56 under submerged fermentation was investigated using agro-industrial wastes and commercial carbon and nitrogen sources. The maximum PGase concentration was equivalent to 8.6 U/mL that was obtained in presence of citrus pectin at 150 rpm, 30 °C, pH = 5.5, and 60 h of fermentation conditions. However, a significant amount of enzyme production was also recorded upon the utilization of corncob (5.3 U/mL) and wheat bran (4.4 U/mL) as carbon sources. Amongst the different nitrogen sources, the highest enzyme production (8.2 U/mL) was obtained in presence of ammonium sulphate and yeast extract simultaneously at a ratio of 1:1. The enzyme was partially purified by gel filtration using Sephadex G50 equilibrated and washed with 50 mM-sodium acetate buffer. The obtained yield and specific activity were determined equivalent to 17% and 9.53 U/mg, respectively. The molecular weight of the partially purified enzyme was estimated as 54 kDa on SDS-PAGE. The conditions affecting the enzyme activity were determined and the highest enzyme activity was recorded at 40 °C and 4.5 pH. Amongst the tested metal ions, 2 and 5 mM of CaCl2 concentrations increased the enzymatic activity by 30%. Overall, the use of corncob (2.5%) to produce PGase by A. pullulans represents an attractive agro-industrial substrate.