The effects of chlorogenic acid (CGA) on growth performance, intestinal morphology, antioxidant capacity, and the autophagy-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in oxidatively stressed broilers were investigated. A total of 400 one-day-old male Cobb broilers were divided randomly into 4 groups using a 2 × 2 factorial arrangement with 2 CGA supplemental levels (0 and 500 mg/kg) and 2 dexamethasone (DEX) challenge levels (0 and 3 mg/kg body weight). All the broilers were injected intraperitoneally with DEX or sterile saline beginning at the age of 15 d for 6 consecutive days. The experiment lasted for 21 d. The CGA increased average daily gain (ADG), villus height, villus height/crypt depth (V/C) value, and the protein expressions of Occludin and ZO-1 in the ileum and decreased the feed:gain (F:G) ratio, which were impaired by the DEX challenge. Superoxide dismutase (SOD), catalase (CAT), gutathione S-transferase (GST), and heme oxygenase-1 (HO-1) activities in the serum and ileum were increased by CGA, whereas protein carboxyl (PCO) level in the serum and ileum, and malondialdehyde (MDA) level in the ileum were decreased of the DEX challenged broilers. The DEX challenge decreased microtubule-associated protein 1 light chain 3 ( LC3 )-II, Beclin1, and autophagy-related gene ( ATG ) 7 mRNA expressions, and the LC3-II/LC3-I value and increased LC3-I, cysteinyl aspartate specific proteinase ( Caspase )-3 and Caspase-9 mRNA expressions in the ileum, which were improved by CGA. DEX also decreased the protein expressions of Kelch-like ECH-associated protein-1 (Keap1), Nrf2, HO-1, NADPH quinone oxidoreductase-1(NQO-1) and increased sequestosome 1 (p62) in the ileum, which were improved by CGA. Interactions occurred between DEX and CGA for the ADG, F:G ratio, villus height, crypt depth, V/C value, and SOD, CAT, GST, and HO-1 activities, MDA and PCO levels, LC3-II/LC3-I value, and expressions of LC3-I, LC3-II, Beclin1, ATG7, Caspase-3, Caspase-9, Occludin, ZO-1, Keap1, Nrf2, HO-1, NQO-1, and p62. In conclusion, CGA improved the growth performance and intestinal health of oxidatively stressed broilers by activating the autophagy-mediated Nrf2 pathway.

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.

Cytochalasans, with diverse structures and pharmacological activities, are a class of compounds containing isoindolinone moieties fused to the tricyclic or tetracyclic ring system. Chaetoglobosin A (cheA), mainly produced by Chaetomium globosum, is the most abundant cytochalasan. However, limited understanding of transcriptional regulation of morphological development and cheA biosynthesis in C. globosum has hindered cheA application in agriculture and biomedical field. This study examined the regulatory role of CgVeA gene in C. globosum. CgVeA had significant effect on secondary metabolites production in C. globosum, similar to that reported in other filamentous fungi. Inactivation of CgVeA caused an obvious decrease in cheA production from 51.32 to 19.76 mg/L under dark conditions. In contrast, CgVeA overexpression resulted in a dramatic increase in cheA production, reaching 206.59 mg/L under light conditions, which was higher than that noted under dark condition. The RT-qPCR results confirmed that CgVeA, as a light responsive regulator, positively regulated cheA biosynthesis by controlling the expression of core genes of the cheA biosynthetic gene cluster and other relevant regulators. Electrophoretic mobility shift assays proved that CgVeA directly regulated LaeA, cheR, and p450, and indirectly regulated PKS. Moreover, CgVeA had a significant effect on the regulation of asexual spores production. When compared with wild-type C. globosum, CgVeA-silenced and CgVeA overexpression mutants presented remarkable differences in sporulation, irrespective of light or dark condition. Besides, CgVeA expression was speculated to negatively regulate spore formation. These findings illustrated the regulatory mechanism of a hypothetical global regulator, CgVeA, in C. globosum, suggesting its potential application in industrial-scale cheA biosynthesis.

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.