The Protein Response of Salt-Tolerant Zygosaccharomyces rouxii to High-Temperature Stress during the Lag Phase

Abstract

Zygosaccharomyces rouxii used in soy sauce brewing is an osmotolerant and halotolerant yeast, but it is not tolerant to high temperatures and the underlying mechanisms remain poorly understood. Using a synthetic medium containing only Pro as a nitrogen source, the response of Z. rouxii in protein level to high-temperature stress (40 °C, HTS) during the lag phase was investigated. Within the first two h, the total intracellular protein concentration was significantly decreased from 220.99 ± 6.58 μg/mg DCW to 152.63 ± 10.49 μg/mg DCW. The analysis of the amino acid composition of the total protein through vacuum proteolysis technology and HPLC showed that new amino acids (Thr, Tyr, Ser, and His) were added to newborn protein over time during the lag phase under HTS. The nutritional conditions used in this study determined that the main source of amino acid supply for protein synthesis was through amino acid biosynthesis and ubiquitination-mediated protein degradation. Differential expression analysis of the amino acid biosynthesis-related genes in the transcriptome showed that most genes were upregulated under HTS, excluding ARO8, which was consistently repressed during the lag phase. RT-qPCR results showed that high-temperature stress significantly increased the upregulation of proteolysis genes, especially PSH1 (E3 ubiquitin ligase) by 13.23 ± 1.44 fold (p < 0.0001) within 4 h. Overall, these results indicated that Z. rouxii adapt to prolonged high temperatures stress by altering its basal protein composition. This protein renewal was related to the regulation of proteolysis and the biosynthesis of amino acids.

Keywords: Zygosaccharomyces rouxii; amino acid biosynthesis; amino acid composition of proteins; high temperature; ubiquitin–proteasome system.

Figure 1

Growth curve analysis of Z. rouxii under two temperatures. Data are presented as the mean ± SEM from three independent experiments.

Figure 2

Trend of total protein content and distribution of amino acid (AA-P) percentage of lag phase. (A) First 12 h total protein concentration in the Z. rouxii cells, (B) initial amino acid composition of the protein (0 h), (C) proteolysis amino acid composition under control during the lag phase (2–4 h), (D) proteolysis amino acid composition under HTS during the lag phase (2–4 h). Data (A) were presented as the mean  ±  SEM from three independent experiments. Different symbols (ns-****) represent significant differences between the two conditions within the same study hours (p < 0.05). Data (B–D) were presented as the mean from three independent experiments.

Figure 3

Consumption of added amino acids and the percentage of free amino acid (AA-F) during lag phase. (A) The remaining Pro concentration in the synthetic medium for the first 12 h, (B) free amino acid composition (except Pro) under control during the lag phase (2 h–4 h), (C) free amino acid composition under HTS during the lag phase (2 h–4 h). Data (A) were presented as the mean ± SEM from three independent experiments. Different symbols (ns-***) represent significant differences between the two conditions within the same study hours (p < 0.05). Data (B,C) were presented as the mean from three independent experiments.

Figure 4

Amino acid biosynthesis pathway map and its gene transcription level expression results of lag phase under HTS. (A) Biosynthesis of amino acids—Zygosaccharomyces rouxii from KEGG (zro01230). (B) Heatmap of transcriptome differentially expression results of under HTS during the lag phase (0 h–2 h–4 h) amino acid biosynthesis pathways genes corresponding to KEGG—Z. rouxii (zro01230). RSEM: TPM, Log2FC > 2 and FDR-p-value < 0.05. Data were presented as three independent experiments.

Figure 5

Key genes for Phe, Tyr, and Lys biosynthesis pathways according to KEGG—Z. rouxii and validation of RT-qPCR levels of amino acids biosynthesis gene downregulated by transcriptional under HTS, (A) Tyr biosynthesis pathway (zro_M00025), (B) Phe biosynthesis pathway (zro_M00024), (C) Lys biosynthesis pathway (zro_M00030), (D) ARO8 RT-qPCR analysis under high-temperature stress during the lag phase (0 h–2 h–4 h). Data (D) were presented as the mean  ±  SEM from three independent experiments. One-way ANOVA with 0 h as reference. Different symbols (*) represent the significant differences between hours within the same study conditions (p < 0.05).

Figure 6

Time course analysis inside 2 conditions 8 proteolysis genes RT-qPCR results: (A) control during the lag phase (0 h–2 h–4 h); (B) HTS during the lag phase (0 h–2 h–4 h). Data were presented as the mean  ±  SEM from three independent experiments. One-way ANOVA with 0 h as reference. Different symbols (*, **, *** and ****) represent significant differences between hours within the same study conditions (p < 0.05).

Figure 7

Lys, Tyr, or Phe was added to the 40 °C 300 g/L glucose condition to determine yeast cell density (OD₆₀₀) at 120 h. CK stands for 40 °C 300 g/L glucose condition. Data were presented as the mean ± SEM from three independent experiments. One-way ANOVA with CK as reference. Different symbols (***) represent the significant differences between amino acids within the same study conditions (p < 0.05).