The ability of the fungus to regulate metabolism on various nitrogen sources makes it survive and metabolize in different environments. The biomass and the β-glucan yield of Aureobasidium pullulans are closely associated with the nitrogen source. This study found the only GATA nitrogen source activation regulating factor Area in HIT-LCY³. In order to testify the Area function, we amplified its conserved domain to build a silencing vector and used the RNAi to obtain the Area silent strain, and then explored its effect on the phenotype of A. pullulans and the yield of β-glucan. We found that the biomass and β-glucan yield of the silent strain decreased significantly after culturing with different nitrogen sources, in particular when using sodium nitrate and glutamate as the source. However, the β-glucan yield increased significantly after overexpression of Area, reaching 5.2 g/L when glutamine was the nitrogen source. In addition, the strain morphology changed as well under different nitrogen sources. At last, we investigated the antioxidant activity in vitro of β-glucan and found that it has a significant clearance effect on OH·, DPPH·, and ABTS·, being best with ABTS. Therefore, this study believed that the Area gene has a certain regulation function on the synthesis of β-glucan with antioxidant activity.

Aureobasidium pullulans (A. pullulans), a commonly found yeast-like fungus, exhibits adaptability to a wide range of pH environments. However, the specific mechanisms and regulatory pathways through which A. pullulans respond to external pH remain to be fully understood. In this study, we first sequenced the whole genome of A. pullulans using Nanopore technology and generated a circle map. Subsequently, we explored the biomass, pullulan production, melanin production, and polymalic acid production of A. pullulans when cultivated at different pH levels. We selected pH 4.0, pH 7.0, and pH 10.0 to represent acidic, neutral, and alkaline environments, respectively, and examined the morphological characteristics of A. pullulans using SEM and TEM. Our observations revealed that A. pullulans predominantly exhibited hyphal growth with thicker cell walls under acidic conditions. In neutral environments, it primarily displayed thick-walled spores and yeast-like cells, while in alkaline conditions, it mainly assumed an elongated yeast-like cell morphology. Additionally, transcriptome analysis unveiled that A. pullulans orchestrates its response to shifts in environmental pH by modulating its cellular morphology and the expression of genes involved in pullulan, melanin, and polymalic acid synthesis. This research enhances the understanding of how A. pullulans regulates itself in diverse pH settings and offers valuable guidance for developing and applying engineered strains.

We have successfully identified the transcription factor Cmr1 from the fungus Aureobasidium pullulans Hit-lcy3T, which regulates melanin biosynthesis genes. Bioinformatics analysis revealed that the Cmr1 gene encodes a protein of 945 amino acids, containing two Cys₂His₂ zinc finger domains and a Zn(II)₂Cys₆ binuclear cluster domain located at the N-terminus of Cmr1. To investigate the function of the Cmr1 gene, we performed gene knockout and overexpression experiments. Our results showed that Cmr1 is a key regulator of melanin synthesis in Hit-lcy3^T, and its absence caused developmental defects. Conversely, overexpression of Cmr1 significantly increased the number of chlamydospores in Hit-lcy3^T and improved melanin production. RT-qPCR analysis further revealed that overexpression of Cmr1 enhanced the expression of several genes involved in melanin biosynthesis, including Cmr1, PKS, SCD1, and THR1. Melanin extracted from the Hit-lcy3^T was characterized using UV and IR spectroscopy. Furthermore, we assessed the antioxidant properties of Hit-lcy3^T melanin and found that it possesses strong scavenging activity against DPPH·, ABTS·, and OH·, but weaker activity against O₂⁻·. These findings suggest that Hit-lcy3^T melanin holds promise for future development as a functional food additive.

Pullulan is a high-value polysaccharide produced through the fermentation of Aureobasidium pullulans. It has significant applications in the fields of food, medicine, environmental science, and packaging. However, the yield, molecular weight, and other characteristics of pullulan can vary depending on the fermentation substrate used. Therefore, it is crucial to analyze the underlying causes of these variations at the molecular level. In this study, we first investigated the morphological differences in A. pullulans YQ65 when cultured in YPD and PDA media. The results indicated that different culture media significantly influence the primary cell morphology of A. pullulans YQ65, which in turn affects the synthesis of secondary metabolites. Subsequently, we employed different culture media to ferment pullulan and examined the variations in pullulan yield, molecular weight, and biomass. Moreover, FTIR and thermodynamic stability tests were conducted to analyze the differences among pullulans across different culture media. Finally, transcriptome analysis revealed that A. pullulans YQ65, when cultured in YPD and PDA media, regulates its growth and metabolism through the expression of key genes that are involved in pathways such as the proteasome, oxidative phosphorylation, metabolism of various secondary metabolites, fatty acid anabolism, carbon metabolism, and amino acid metabolism. The transcriptome results were further validated by assessing the expression of specific genes. This study enhances the understanding of the fermentation differences observed with different substrates in A. pullulans and provides valuable insights for optimizing culture substrates. Additionally, it offers guidance for utilizing agricultural and forestry processing waste, as well as food processing by-products, to produce pullulan cost-effectively in the future.

Chaetoglobosin A (CheA), a well-known macrocyclic alkaloid with prominently highly antimycotic, antiparasitic, and antitumor properties, is mainly produced by Chaetomium globosum. However, a limited understanding of the transcriptional regulation of CheA biosynthesis has hampered its application and commercialization in agriculture and biomedicine. Here, a comprehensive study of the CgXpp1 gene, which encodes a basic helix-loop-helix family regulator with a putative role in the regulation of fungal growth and CheA biosynthesis, was performed by employing CgXpp1-disruption and CgXpp1-complementation strategies in the biocontrol species C. globosum. The results suggest that the CgXpp1 gene could be an indirect negative regulator in CheA production. Interestingly, knockout of CgXpp1 considerably increased the transcription levels of key genes and related regulatory factors associated with the CheA biosynthetic. Disruption of CgXpp1 led to a significant reduction in spore production and attenuation of cell development, which was consistent with metabolome analysis results. Taken together, an in-depth analysis of pleiotropic regulation influenced by transcription factors could provide insights into the unexplored metabolic mechanisms associated with primary and secondary metabolite production.

Chaetoglobosin A (CheA), a typical structure of the cytochalasin family, exhibits outstanding efficacy against a variety of tumor cells and plant pathogens. However, its low yield and high production cost are major obstacles limiting its wide application. In order to increase CheA yield, an engineered strain was established by overexpressing CgMfs, the gene encoding the MFS family’s efflux pump, on chassis cells lacking CgXpp1, which have been shown to act as a negative regulator of CheA biosynthesis. As expected, the engineered strain significantly boosted CheA production from 63.19 to 265.93 mg/L after incubation in PDA medium for 10 d, whereas the yield of the engineered strain was remarkably enhanced 2.93-fold compared with the wild type, following 10 d of cultivation utilizing potato starch industrial waste. The addition of metal ions had a positive effect on CheA production, with Cu²⁺ being the most effective and improving production to 176.92 mg/L. The optimal fermentation conditions were determined by response surface optimization, and under the optimal conditions, the engineered strain could stably produce CheA with a yield of 197.58 mg/L. This study provided the conditions for reducing production costs while increasing CheA production, as well as new strategies and insights for the production of the target compound.