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.

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.

Natronorubrum daqingense JX313^T is an extremely halophilic archaea that can grow in a NaCl-saturated environment. The excellent salt tolerance of N. daqingense makes it a high-potential candidate for researching the salt stress mechanisms of halophilic microorganisms from Natronorubrum. In this study, transcriptome analysis revealed that three genes related to the biosynthesis of vitamin B₁₂ were upregulated in response to salt stress. For the wild-type (WT) strain JX313^T, the low-salt adaptive mutant LND5, and the vitamin B₁₂ synthesis-deficient strain ΔcobC, the exogenous addition of 10 mg/L of vitamin B₁₂ could maximize their cell survival and biomass in both optimal and salt stress environments. Knockout of cobC resulted in changes in the growth boundary of the strain, as well as a significant decrease in cell survival and biomass, and the inability to synthesize vitamin B₁₂. According to the HPLC analysis, when the external NaCl concentration (w/v) increased from 17.5% (optimal) to 22.5% (5% salt stress), the intracellular accumulation of vitamin B₁₂ in WT increased significantly from (11.54 ± 0.44) mg/L to (15.23 ± 0.20) mg/L. In summary, N. daqingense is capable of absorbing or synthesizing vitamin B₁₂ in response to salt stress, suggesting that vitamin B₁₂ serves as a specific compatible solute effector for N. daqingense during salt stress.