AozC, a zn(II)2Cys6 transcription factor, negatively regulates salt tolerance in Aspergillus oryzae by controlling fatty acid biosynthesis

Abstract

Background: In the soy sauce fermentation industry, Aspergillus oryzae (A. oryzae) plays an essential role and is frequently subjected to high salinity levels, which pose a significant osmotic stress. This environmental challenge necessitates the activation of stress response mechanisms within the fungus. The Zn(II)₂Cys₆ family of transcription factors, known for their zinc binuclear cluster-containing proteins, are key regulators in fungi, modulating various cellular functions such as stress adaptation and metabolic pathways.

Results: Overexpression of AozC decreased growth rates in the presence of salt, while its knockdown enhanced growth, the number of spores, and biomass, particularly under conditions of 15% salt concentration, doubling these metrics compared to the wild type. Conversely, the knockdown of AozC via RNA interference significantly enhanced spore density and dry biomass, particularly under 15% salt stress, where these parameters were markedly improved over the wild type strain. Moreover, the overexpression of AozC led to a downregulation of the FAD2 gene, a pivotal enzyme in the biosynthesis of unsaturated fatty acids (UFAs), which are essential for preserving cell membrane fluidity and integrity under saline conditions. Transcriptome profiling further exposed the influence of AozC on the regulation of UFA biosynthesis and the modulation of critical stress response pathways. Notably, the regulatory role of AozC in the mitogen-activated protein kinase (MAPK) signaling and ABC transporters pathways was highlighted, underscoring its significance in cellular osmotic balance and endoplasmic reticulum homeostasis. These findings collectively indicate that AozC functions as a negative regulator of salt tolerance in A. oryzae.

Conclusion: This research suggest that AozC acts as a negative regulator in salt tolerance and modulates fatty acid biosynthesis in response to osmotic stress. These results provide insights into the regulatory mechanisms of stress adaptation in A. oryzae.

Keywords: Aspergillus oryzae; Differentially expressed genes; Fatty acid; Salt treatment; Transcription factor; Transcriptome; Unsaturated fatty acids.

Fig. 1

Analysis of AozC gene expression and phylogenetic characterization. (A) The relative expression levels of the AozC gene across various salt concentrations (0%, 5%, 10%, and 15%) at different time points (24 h, 48 h, and 72 h) post-treatment. Each bar represents the mean expression level ± standard error (SE) derived from three independent biological replicates. The error bars indicate the variability among the replicates, and statistical significance was assessed using a one-way ANOVA with a Tukey’s post-hoc test for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). (B) The conserved functional domain(s) within the AozC protein, highlighting the key structural features that define its role as a transcription factor in A. oryzae. The Zn cluster region is located at the N-terminus, which is consistent with the N-terminal position of most Zn(II)₂Cys₆ transcription factors. (C) The maximum likelihood method implemented in MEGA 5.0 software was utilized to construct a phylogenetic tree, comparing the AozC protein sequence with homologous sequences from other Aspergillus species. The consensus tree, depicted here, is supported by the results of 1000 bootstrap replications, indicating the reliability of the phylogenetic relationships presented

Fig. 2

Phenotypic and molecular analysis of overexpression of AozC in S. cerevisiae under salt stress. (A-C) The optical density (OD) values of overexpression of AozC (pYES2-AozC) in S. cerevisiae strains were measured after exposure to liquid media with varying salt concentrations at 12 h (A), 24 h (B), and 36 h (C) of growth. (D) The phenotype of WT and overexpression of AozC in S. cerevisiae strains on solid media with different salt concentrations after 36 h of growth. The numerical annotations succeeding the strain identifiers, such as ‘1:1,’ ‘1:10,’ and ‘1:100’, indicate the dilution ratios of the S. cerevisiae cultures. (E) The content of fatty acids in WT and overexpression of AozC in S. cerevisiae strains was determined

Fig. 3

Impact of salt stress on overexpressing and RNAi of AozC in A. oryzae strains. (A) The phenotype of WT and AozC transgenic strains under normal and salt treatment at 72 h post-inoculation (hpi). (B) The phenotype of WT and A. oryzae strains with RNAi targeting the AozC gene under normal and salt treatments at 72 hpi. (C) The spore density for WT, overexpressing and RNAi of AozC in A. oryzae strains were assessed in response to salt treatments. (D) The dry biomass for WT, overexpressing and RNAi of AozC in A. oryzae strains under salt stress conditions. The bars represent the average (± standard error, SE) of three biological repeats, indicating the reproducibility of the results. Asterisks indicate the presence of statistically significant differences between the treatment and the control samples based on Student’s t tests (^ns No significant difference; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001)

Fig. 4

Analysis of FAD2 expression and fatty acid content in WT, overexpression and RNAi of AozC in A. oryzae strains under normal conditions. (A) The relative expression levels of the FAD2 gene in WT, overexpression and RNAi of AozC in A. oryzae strains under normal growth conditions. (B) The intracellular content of saturated or unsaturated fatty acids, as a percentage of the total fatty acid content in WT, overexpression, and RNAi of AozC in A. oryzae under normal growth conditions. Asterisks indicate the presence of statistically significant differences between the treatment and the control samples based on Student’s t tests (^ns No significant difference; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001)

Fig. 5

Venn diagram displaying the distribution of differentially expressed genes among WT, overexpression and RNAi of AozC strains in A. oryzae