The Deletion of LeuRS Revealed Its Important Roles in Osmotic Stress Tolerance, Amino Acid and Sugar Metabolism, and the Reproduction Process of Aspergillus montevidensis

Abstract

Aspergillus montevidensis is an important domesticated fungus that has been applied to produce many traditional fermented foods under high osmotic conditions. However, the detailed mechanisms of tolerance to osmotic stress remain largely unknown. Here, we construct a target-deleted strain (ΔLeuRS) of A. montevidensis and found that the ΔLeuRS mutants grew slowly and suppressed the development of the cleistothecium compared to the wide-type strains (WT) under salt-stressed and non-stressed conditions. Furthermore, differentially expressed genes (p < 0.001) governed by LeuRS were involved in salt tolerance, ABC transporter, amino acid metabolism, sugar metabolism, and the reproduction process. The ΔLeuRS strains compared to WT strains under short- and long-term salinity stress especially altered accumulation levels of metabolites, such as amino acids and derivatives, carbohydrates, organic acids, and fatty acids. This study provides new insights into the underlying mechanisms of salinity tolerance and lays a foundation for flavor improvement of foods fermented with A. montevidensis.

Keywords: Aspergillus montevidensis; leucyl-tRNA synthetase; metabolome; osmotic tolerance; transcriptome.

Figure 1

Morphological characteristics, growth rates, and antioxidative activities of the wide-type A. montevidensis (WT) and ΔLeuRS mutant grown on YPD with and without 1.5 M NaCl at 28 °C for 14 days. Under saline conditions, colony morphology of WT (a) and ΔLeuRS mutant (d), in situ observation of the microscopic morphological characteristics of WT (b) and mutant (e), cleistothecia of WT (c) and conidial heads of ΔLeuRS (f). Colonial characteristics and microscopic characteristics of WT (g,h) and ΔLeuRS (i,j) grown on YPD without NaCl. Growth rates (k) and antioxidant activities (l) of WT and ΔLeuRS mutants (*, p < 0.05, **, p < 0.01, *** p < 0.001, ****, p < 0.0001, ns, p > 0.05).

Figure 2

PCA (a) and Venn analysis of genes up- (b,c) and down-expressed (d,e) in ΔLeuRS mutants and WT strains under different saline conditions (C = control, SS = short-term salt stress, LS = long-term salt stress).

Figure 3

KEGG enrichment analysis of genes up- and down-expressed in ΔLeuRS (a,b) and WT (c,d), respectively (C = control, SS = short-term salt stress, LS = long-term salt stress).

Figure 4

Heat map analysis of relative differential expression levels of some DEGs in compared groups of ΔLeuRS mutants and WT strains under distinct osmotic stress conditions.

Figure 5

Analysis of discriminatory metabolites (p < 0.05, log2FC ≥ 1.0). The relative abundances of compounds identified only in ΔLeuRS_SS/ΔLeuRS_C and WT_SS/WT_C (a) and molecules co-occurred in these two groups (b). The relative contents of the unique compounds detected only in ΔLeuRS_LS/ΔLeuRS_C and WT_LS/WT_C (c) and compounds shared by these two groups (d).

Figure 6

Conceptual profile showing enriched pathways of DEGs and metabolites between ΔLeuRS mutants and WT strains. DEGs involved in sexual and asexual processes (a). DEGs and metabolites were related to the metabolic pathways of amino acids, sugars, organic acids, and fatty acids (b). Genes marked with red or green color showed that these were up- and down-regulated at different time points.