###### Abstract  
Pullulan, which is a microbial exopolysaccharideadride, 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 \(115.4\pm 1.82\) 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. Keywords:pullulan; _AureobasidiumAureobasidium_ spp. strains are well known to be the primary producers of pullulan. The strains are multicellular fungi encompassing yeast-like cells, swollen cells, conidia, chlamydospores, and mycelia types that thrive in diverse habitats, such as soils, plant leaves, oceans, and extreme environments [7; 8; 9]. Among the species of _Aureobasidium_, _A. pullulans_ and _A. melanogaster_ emerge as the most robust pullulan producers [1; 10; 11]. However, most wild-type strains yield less than 78 g/L of pullulan [2; 3; 8; 12]. Only a few wild isolates were found to produce relatively high pullulan titers (>100 g/L). For instance, _A. melanogaster_ A4 using maltose achieved 122.3 g/L pullulan with a yield of 0.4 g/g via fed-batch fermentation and another strain _A. melanogaster_ TN1-2 produced 114 g/L pullulan from sucrose with 0.81 g/g yield via batch fermentation [10; 13]. Some engineered mutants also exhibited distinctly elevated pullulan production, such as the mutant _A. pullulans_ M233-20, which produced 162.3 g/L pullulan [14]. To improve pullulan production, various measures, including condition optimization, mutagenesis, genetic modification, and substance addition, were employed [9; 15; 16]. However, the improvements in pullulan production achieved using these measures were limited to no more than 35%, which, in return, impacted the molecular weight of pullulan [1; 15; 17]. Hence, the above-mentioned measures are still insufficient to meet the requirements of efficient and high-quality industrial pullulan production. On the other hand, high pullulan production requires high sugar concentrations, which increase the osmotic stress and actually make osmophilic or osmotolerant strains more suitable for pullulan production [18]. Most _Aureobasidium_ strains prefer to use sucrose as the best carbon source for pullulan production, while only a small amount of them prefer utilizing glucose, fructose, or maltose [2; 10; 18; 19]. For example, _A. melanogaster_ P16 produced the highest pullulan titer (65.3 g/L) using sucrose in comparison with glucose, fructose, glycerol, and inulin [12].