Proline protects Wickerhamomyces anomalus against ethanol‑induced damage during logarithmic growth: integrated physiological, transcriptomic, and metabolomic analyses

Abstract

Background

Proline plays a critical regulatory role in microbial stress responses; however, its specific mechanism of action under ethanol stress in Wickerhamomyces anomalus during logarithmic growth remains poorly understood.

Results

This study investigated the protective effects of proline against ethanol-induced injury and elucidated the mechanism by which it enhances resistance to 9% (v/v) ethanol stress. The results indicated that proline supplementation significantly increased the viability of ethanol-stressed yeast and stimulated cellular proline transport and catabolism. Furthermore, proline mitigated the impairment in morphology and ultrastructure of yeast cells by maintaining cell wall, cell membrane, and mitochondrial integrity, ameliorating oxidative stress, and restoring cellular homeostasis under ethanol stress. Further analysis revealed that the protective effect of proline on cell membrane homeostasis was related to decreased membrane permeability, increased membrane fluidity, and elevated intracellular K⁺ levels. The alleviation of oxidative stress by proline supplementation was associated with enhanced antioxidant enzyme activities and increased glutathione levels. Transcriptomic and metabolomic analyses indicated that proline regulated ribosome synthesis, the cell wall integrity pathway, ABC transporters, and arginine and proline metabolism.

Conclusions

These findings indicate that proline effectively protects yeast from ethanol stress, supporting the development of ethanol-tolerant W. anomalus strains for industrial fermentation.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12866-026-05373-1.

Background

Wickerhamomyces anomalus has garnered increasing attention within the wine industry in recent years [1, 2]. Owing to its distinctive metabolic properties, this species is frequently isolated from diverse fermentation environments, including Baijiu [3], rice wine [4], and various fruit wines [5–7]. Traditionally, W. anomalus has been recognized as a potent producer of glycosidases—such as β-D-glucosidase, α-L-arabinofuranosidase, α-L-rhamnosidase, and β-D-xylosidase—which play a crucial role in releasing volatile aroma compounds from glycosylated precursors during fermentation [8]. Moreover, its notable capacity to generate elevated levels of acetate esters, which impart desirable floral and fruity notes, has made it a promising starter culture in winemaking practices [8].

Nevertheless, yeast cells face multiple stress factors during wine fermentation, including ethanol, osmotic, and temperature stress [9]. Among these, high ethanol concentration is considered the primary stressor due to its toxicity to yeast cells [10]. It has been reported that ethanol stress disrupts the intracellular redox balance, inducing reactive oxygen species (ROS) bursts and oxidative stress [11]. This, in turn, causes oxidative damage to key biological macromolecules such as proteins, nucleic acids, and lipids. The impairment of these macromolecules compromises the structural integrity of cellular components, including the cell membrane, cell wall, and mitochondria [12]. Consequently, it reduces membrane fluidity and diminishes the capacity for substance transport. Furthermore, ethanol stress can oxidize and denature enzymes responsible for regulating metabolic reactions, leading to metabolic dysregulation. Collectively, these effects inhibit cell growth, increase cell mortality, and ultimately reduce fermentation efficiency [12, 13].

Numerous strategies have been employed to enhance the ethanol tolerance of yeast strains, which generally include genetic engineering and exogenous nutrient supplementation [14, 15]. Among these, exogenous nutrient supplementation—particularly with nitrogen sources—has gained broader acceptance in the field of food fermentation [16]. This preference is due to both safety concerns regarding genetically modified foods and the remarkable ability of such supplements to regulate yeast physiology and metabolism [13, 17]. Recent studies indicate that amino acids such as tryptophan [18] and arginine play a role in the ethanol stress response of yeast cells [19–21]. Exogenous supplementation of these amino acids enhances cellular structural integrity, stabilizes intracellular homeostasis, and promotes cell growth [20]. In our previous study on W. anomalus, integrated transcriptomic and metabolomic analyses further demonstrated that ethanol stress markedly alters the expression of genes and the accumulation of metabolites within proline-related pathways [22]. These results indicate that proline and its interconnected metabolism play critical regulatory roles in the ethanol stress response of W. anomalus.

Proline is a nonpolar aliphatic amino acid that participates in protein synthesis and contributes to the formation of various structural and functional molecules, such as membrane components, enzymes, and carrier receptors [23]. More importantly, proline plays a crucial regulatory role in the stress responses of plants [24] and microorganisms [25]. For instance, it has been reported that Bacillus megaterium produces and secretes proline into the extracellular medium, thereby conferring acid tolerance [26]. In yeast species, proline is involved in regulatory responses to diverse stresses, including temperature, acid, osmotic, ethanol, and furan stress [23]. Accumulating evidence indicates that proline acts as a key metabolic regulator in mediating S. cerevisiae’s adaptive response to ethanol-induced stress. Although ethanol stress does not trigger rapid proline accumulation in S. cerevisiae, proline levels increase significantly—up to twofold—following ethanol exposure [27]. Moreover, mutants with altered proline metabolism exhibit corresponding changes in ethanol tolerance [28]. Further studies have shown that proline’s ability to alleviate ethanol stress in S. cerevisiae is mechanistically associated with its efficient scavenging of ROS. Mutants that accumulate high proline concentrations show reduced ROS levels under ethanol stress and demonstrate improved cell viability [29]. Given the established role of proline in yeast stress responses [23] and preliminary omics data from our previous study [22], it is speculated that proline supplementation could enhance the stress tolerance of W. anomalus. However, the specific mechanism by which proline modulates the ethanol stress response in W. anomalus remains to be elucidated.

Therefore, this study aimed to investigate the effects of proline on the cell viability, membrane and wall integrity, and cellular redox homeostasis of W. anomalus under ethanol stress. Furthermore, the potential mechanism for improved ethanol stress tolerances in W. anomalus with proline supplementation was also elucidated by integrating transcriptomic and metabolomic approaches. These findings provide a theoretical basis for the enhancement of the ethanol stress resistance of W. anomalus.

Results and discussion

Proline supplementation enhanced the ethanol tolerance of W. anomalus

To evaluate the impact of proline supplementation on the viability of yeast under ethanol stress, cell survival, death, and biomass were assessed using spot assay, methylene blue staining, and dry weight assay, respectively. As shown in Fig. 1A-B, at the beginning of the treatment, cell survival and death rates were similar across all groups, with no significant differences observed. However, after ethanol stress treatment, cell survival was markedly inhibited, and the death rate increased significantly (3 ± 0.4% in the control group vs. 31 ± 3% in the ethanol group). In contrast, compared with the ethanol group, proline supplementation notably improved cell survival and reduced the death rate (23 ± 2% in the Eth + Pro group vs. 31 ± 3% in the ethanol group). A similar beneficial effect of proline supplementation was also reflected in the biomass of ethanol-stressed yeast cells, as measured by dry weight (Fig. 1C). These results indicate that proline addition partially reversed the ethanol-induced reduction in cell viability of W. anomalus, thereby enhancing the ethanol tolerance of the yeast cells. Previous studies have confirmed that S. cerevisiae proline metabolism mutants (such as GK^I150T, GK^D154N, GK^1P247S, and GK^E415K), which accumulate higher intracellular proline concentrations, exhibit enhanced ethanol tolerance [23]. Therefore, it is reasonable to speculate that exogenous proline supplementation may facilitate endogenous accumulation and utilization, ultimately improving the ethanol tolerance of W. anomalus.

Fig. 1.

Effect of proline on cell viability, proline transporter expression, and the expression of key catabolic enzymes in W. anomalus under ethanol stress. The ethanol- stressed yeast cells were treated with 9% ethanol (v/v). The concentration of exogenous proline was 50 mmol/L. A Yeast viability assessed by spot dilution assay. B Cell death rate. C Biomass of yeast cells. D Schematic diagram of proline metabolic pathway in yeast. Red marks indicate the proline anabolic pathway in the cytoplasm, while blue marks are the proline catabolic pathways in mitochondria. E Relative expression of the GAP1 gene. F Relative expression of the PUT4 gene. G Relative expression of the PUT1 gene. H Relative expression of the PUT2 gene. a, b, c significant differences between control and samples groups at p < 0.05

To test this hypothesis, we analyzed the expression levels of proline transporters and key catabolic enzymes (Fig. 1D) using RT-qPCR. Compared with the control group, the expression of both GAP1 and PUT4 was down-regulated in the ethanol group. However, under ethanol stress with simultaneous proline addition, GAP1 expression was significantly up-regulated by 3.8-fold, and PUT4 expression was up-regulated by 20-fold (Fig. 1E-F). It has been reported that proline can be transported across the membrane via the general amino acid permease (GAP1) and the high-affinity proline-specific transporter (PUT4) [30, 31]. The decreased expression of GAP1 and PUT4 under ethanol stress suggests that ethanol impairs proline transmembrane transport, likely due to reduced membrane fluidity and permeability. The up-regulation of both transporters upon proline addition indicates that exogenous proline promotes intracellular proline uptake, mediated by GAP1 and PUT4. To further verify this, we measured the proline content in W. anomalus supplemented with exogenous proline. As expected, endogenous proline levels were significantly increased following this supplementation (Supplementary Fig. 1).

Proline degradation is catalyzed by proline oxidase (PO, EC 1.4.3.2, encoded by PUT1) and P5C dehydrogenase (P5CD, EC 1.5.1.12, encoded by PUT2) within mitochondria [32]. We further examined the expression of PUT1 and PUT2 and found that ethanol stress reduced their expression by 7.7-fold and 1.85-fold, respectively (Fig. 1G-H). As expected, when proline was supplied under ethanol stress, the expression of PUT1 and PUT2 was significantly enhanced by 5.23-fold and 3.28-fold, respectively. These data suggest that proline supplementation enhances proline catabolism, which may contribute to the improved ethanol tolerance observed in W. anomalus.

Proline supplementation mitigated the alterations in morphology and ultrastructure of W. anomalus against ethanol challenge

Electron microscopy can clearly reveal morphological and structural alterations in yeast cells under various environmental stress conditions, as it enables detailed observation of critical ultrastructural features such as the cell wall, plasma membrane, and organelle morphology and integrity [33, 34]. In this study, we employed scanning and transmission electron microscopy to investigate how ethanol stress damages W. anomalus cells and to evaluate the protective role of exogenous proline.

In the absence of ethanol, cells exhibited a plump, elliptical morphology with smooth surfaces (Fig. 2A). Under ethanol stress, however, many cells showed significant morphological alterations, including swelling and noticeably increased surface roughness (Fig. 2B). In contrast, proline supplementation alleviated these ethanol-induced morphological changes, although some cells still appeared swollen (Fig. 2C). Furthermore, TEM observations of yeast ultrastructure revealed that normal cells possessed intact cell walls, cytoplasmic membranes, nuclei, and mitochondria. Ethanol stress, however, led to thinner and coarser cell walls, along with visible damage to the cytoplasmic membrane (Fig. 2D). Nuclear disintegration was also observed, and mitochondria became swollen, with cristae appearing dispersed and disorganized (Fig. 2E). After proline treatment, the cell walls, membranes, nuclei, and mitochondria appeared more intact, although mitochondrial swelling persisted in some cells (Fig. 2F).

Fig. 2.

Effect of proline on the morphological characteristics of W. anomalus under ethanol stress. A–C Representative SEM images of cells from the control (A), ethanol-treated (B), and ethanol + proline (C) groups. Red arrows highlight surface irregularities indicating cell swelling or increased roughness. (D–F) Representative TEM images of cells from the control (D), ethanol-treated (E), and ethanol + proline (F) groups. Red arrows indicate regions of cell wall thinning or membrane damage. Scale bars: 5 μm (A–C); 1 μm (D–F). Abbreviations: CM, cytoplasmic membrane; CW, cell wall; M, mitochondria; N, nucleus

These results indicate that ethanol stress compromises the structural integrity of the cell wall, membrane, nucleus, and mitochondria in W. anomalus, thereby altering its morphology. The observed impairment of these structures likely contributes to the loss of cellular function and ultimately to cell death, underscoring the importance of structural integrity for yeast survival under stress. Proline supplementation, however, improved the integrity of these damaged structures, which may explain its role in enhancing cell viability (Fig. 1A-C). Similar protective effects have been reported for other exogenous nutrients—including amino acids, peptides, protein hydrolysates, and metal ions—which have been shown to mitigate ethanol-induced damage to the cell wall, cell membrane, and intracellular organelles [12, 35].

Proline supplementation ameliorates the disruption of cell wall homeostasis in W. anomalus against ethanol challenge

The cell wall is crucial for maintaining yeast cell integrity, as it provides structural strength, defines cell shape, and protects against environmental stress [36]. Therefore, maintaining cell wall homeostasis represents a key strategy for enhancing cellular stress tolerance. Given the observed improvements in cell wall morphology under electron microscopy, we further investigated cell wall homeostasis in W. anomalus by analyzing its composition and function.

As shown in Fig. 3A, the content of β-1,3-glucan—the fundamental structural framework of the cell wall—was quantified. Under ethanol stress, β-1,3-glucan content increased significantly, reaching approximately 13% that of the control. When proline was supplemented along with ethanol stress, the β-1,3-glucan content further increased by 23% compared to the ethanol group. Chitin, a linear polymer of β-1,4-linked N-acetylglucosamine, forms the inner layer of the cell wall together with β-glucan [37]. In our study, ethanol stress led to a significant 21.7% increase in chitin content in W. anomalus cells compared to the control(Fig. 3B). Furthermore, supplementation with proline under ethanol stress (Eth + pro group) resulted in an additional elevation, with chitin content being 69.8% higher than that in the ethanol-stressed group without proline.

Fig. 3.

Effect of proline on the cell wall homeostasis of W. anomalus under ethanol stress. A The β-1,3-glucan content. B The chitin content. C The sensitivity to lyticase. D Extracellular protein leakage. a, b, c significant differences between control and samples groups at p < 0.05

Previous research indicates that exogenous proline can regulate the expression of key genes involved in the synthesis of cellulose, hemicellulose, lignin, and pectin, and can modulate pectin methylesterase activity in plant cell walls under metal stress, thereby alleviating heavy metal toxicity [38]. This suggests that proline may enhance stress tolerance partly by remodeling cell wall structure and function. In the present study, the addition of proline enhanced the synthesis of both β-1,3-glucan and chitin, indicating that proline promotes structural remodeling of the cell wall in W. anomalus under ethanol stress.

Lyticase is an enzyme complex that primarily hydrolyzes β-1,3-glucan, a major structural component of the yeast cell wall, and is routinely used to assess the integrity of the glucan layer [14, 35]. Under ethanol stress, we evaluated the susceptibility of W. anomalus cell walls to lyticase. As shown in Fig. 3C, cells exposed to ethanol for 0–70 min exhibited significantly increased lyticase sensitivity, indicating compromised cell wall integrity (CWI). The addition of proline reduced this ethanol-induced susceptibility to lyticase. In addition, quantitative analysis of the lyticase sensitivity curves (Fig. 3C) revealed a linear decrease in relative cell viability over time for all treatments (R² ≥ 0.977). The slope of the Ethanol group (–0.514 h⁻¹) was steeper than that of the Control (–0.380 h⁻¹) and the Eth + Pro group (–0.461 h⁻¹), indicating that ethanol accelerated cell wall degradation, while proline supplementation partially alleviated this effect.

Furthermore, the effect of ethanol on CWI was assessed by measuring the release of intracellular proteins due to cell lysis [35]. Ethanol stress significantly increased protein leakage compared to the control, suggesting cell wall damage (Fig. 3D), while proline supplementation reduced this leakage. Taken together, ethanol stress induced cell wall damage and impaired its integrity. Proline supplementation partially counteracted these detrimental effects, thereby helping to maintain cell wall integrity and overall homeostasis.

Proline supplementation restores the disruption of cell membrane homeostasis in W. anomalus against ethanol challenge

Cell wall modifications inevitably affect the function of the cell membrane in resisting various environmental stresses [39]. As a natural barrier, the cell membrane forms a protective boundary between the cell and its external environment by maintaining structural integrity and selective permeability. This thin, flexible lipid bilayer serves as a dynamic shield that regulates the passage of substances while excluding harmful agents [40]. Its capacity to sustain this protective role is crucial for cellular homeostasis, enabling the cell to survive and function under diverse and often harsh conditions [41].

To evaluate the impact of ethanol stress on the cell membrane of W. anomalus, we assessed its integrity, permeability, and fluidity. As shown in Fig. 4A–B, nearly all cells in the control group were stained green, indicating intact membranes. Under ethanol stress, however, the number of green-stained cells decreased significantly, suggesting impaired membrane integrity. In contrast, the Eth + pro group showed a significantly higher count of green-stained cells compared to the ethanol-only group, indicating that exogenous proline improved membrane integrity. Similar results were obtained using propidium iodide (PI) staining. A significantly higher proportion of cells exhibited red fluorescence after ethanol treatment, whereas simultaneous addition of proline with ethanol significantly reduced the proportion of PI-positive cells (Fig. 4C–D).

Fig. 4.

Effect of proline on the cell membrane homeostasis of W. anomalus under ethanol stress. (A, B) FDA staining for membrane integrity. (C, D) PI staining for membrane integrity. (E) Membrane permeability via relative electrical conductivity assay. (F) Membrane fluidity represented by the generalized polarization (GP) value. (G) Contents of intracellular K+. Scale bars: A = 300 µm. a, b, c significant differences between control and samples groups at p<0.05

Ethanol-induced disruption of membrane homeostasis was further examined by measuring relative electrical conductivity [42]. As shown in Fig. 4E, ethanol stress significantly increased membrane permeability, elevating relative electrical conductivity by 24.54% compared to the control. Supplementation with proline significantly reduced this value by 12.73% relative to the ethanol-stressed group. Membrane fluidity, a key parameter of membrane homeostasis, plays a vital role in stress adaptation and normal physiological function. It was evaluated using the generalized polarization (GP) value, where a higher GP indicates lower fluidity [35]. Ethanol stress increased the GP value by approximately 5.59-fold compared to the control, reflecting markedly reduced membrane fluidity (Fig. 4F). The addition of proline significantly lowered the GP value relative to the ethanol-only group. Previous studies have suggested that reduced membrane fluidity can enhance ethanol tolerance [43]. Consistent with this, our findings indicate that proline supplementation decreases membrane fluidity in W. anomalus cells. This adjustment likely helps maintain membrane homeostasis, thereby contributing to enhanced resistance against ethanol stress. A similar protective role of proline has been reported in Lacticaseibacillus rhamnosus under hyperosmotic stress, where it reduces the leakage of intracellular metabolic enzymes and stabilizes the cell membrane structure, ultimately improving bacterial survival.

Elevated intracellular K⁺ levels are critical for yeast survival, regulating cell volume, plasma membrane potential, and metabolic activity [44]. K⁺ also serves an essential function in how plants and yeast respond to diverse environmental stressors [42, 45]. Supporting this, exogenous K⁺ has been found to improve the tolerance of S. pastorianus to both osmotic and ethanol stress through the restoration of membrane function [46]. In this study, we measured intracellular K⁺ levels in W. anomalus under ethanol stress. Figure 4G shows that proline addition led to a significant rise in intracellular K⁺ (196.49 mmol/L), reaching a level 2.04-fold higher than that in the ethanol-stressed group (96.54 mmol/L). This observation supports prior findings that sustaining high cytosolic K⁺ concentrations enhances ethanol tolerance in S. cerevisiae [47, 48].

Proline supplementation restores mitochondrial integrity and ATP synthesis in W. anomalus against ethanol challenge

Mitochondria serve as central hubs of cellular energy metabolism and play critical roles in stress response [49]. Structurally, mitochondrial integrity is essential for maintaining electron transport and enabling efficient ATP synthesis via oxidative phosphorylation [50]. Under environmental stress, however, mitochondrial structure is often disrupted and its function compromised. The effects of proline supplementation on mitochondrial integrity and function in W. anomalus under ethanol stress were evaluated by measuring mitochondrial membrane potential (ΔΨm) and intracellular ATP concentration. Fluorescence-based assessment of ΔΨm revealed that ethanol stress significantly depolarized mitochondria, reducing ΔΨm by approximately 65% (Fig. 5A–B). In contrast, proline supplementation under ethanol stress significantly increased ΔΨm by 19.23% relative to the ethanol-treated group, suggesting that proline contributes to the restoration of mitochondrial integrity.

Fig. 5.

Effect of proline on mitochondrial integrity and function of W. anomalus under ethanol stress. A-B Mitochondrial membrane potential measured by rhodamine 123 assay. C Intracellular ATP content. Scale bars: A = 300 μm. a, b, c significant differences between control and samples groups at p < 0.05

Since membrane damage can inhibit ATP synthase activity and reduce ATP output, intracellular ATP levels were further assessed to evaluate mitochondrial function. Ethanol stress markedly decreased ATP content to 34.94% of the control level (Fig. 5C). Proline supplementation reversed this inhibition, raising ATP levels approximately 8.65-fold compared to the ethanol-stressed group, which suggests that proline treatment restores ATP synthesis. Similar protective effects of proline in restoring mitochondrial integrity and ATP synthesis have been widely reported in senescent animal cells [51] and stressed plant cells [52]. Notably, ATP content in the Eth + Pro group also exceeded that of the control. This may be explained by the dual role of proline: in addition to acting as a protectant, it can also serve as a nutrient that participates in energy synthesis through carbon–nitrogen metabolic pathways.

Proline supplementation ameliorates oxidative stress in W. anomalus against ethanol challenge

Mitochondria, specifically the mitochondrial electron transport chain (mtETC), are recognized as a primary site of reactive oxygen species (ROS) generation [20, 40]. Excessive ROS production can induce oxidative stress and lead to oxidative damage to biological macromolecules and cellular structures, including the cell wall and membranes [12]. Given the observed impairment of mitochondrial integrity and function under ethanol stress—and the subsequent improvement with proline supplementation—we assessed ROS levels in W. anomalus using fluorescence staining. ROS were significantly elevated after ethanol treatment compared to the control (Fig. 6A–D). In contrast, proline supplementation under ethanol stress markedly reduced ROS accumulation. It should be noted, however, that the ROS detection methods employed have certain limitations. While DCFH‑DA and DHE—used in this study and widely adopted as classic fluorescent ROS probes—provide a general indication of oxidative status, they are susceptible to non‑specific reactions. For instance, DCFH‑DA fluorescence can be influenced by elevated metal ion concentrations and peroxidase activity [53]. Although more specific alternatives, such as boronate-based or genetically encoded probes, are increasingly recognized, their commercial availability and practical application remain limited.

Fig. 6.

The impact of proline on ethanol-induced oxidative stress in W. anomalus. A, B Intracellular H₂O₂ levels measured using the fluorescent probe DCFH-DA. C, D Intracellular superoxide anion (O₂•⁻) levels measured using the fluorescent probe DHE. E MDA content. F SOD activity. G CAT activity. H GSH content. Scale bar = 300 μm. a, b, c significant differences between control and samples groups at p < 0.05

Furthermore, under ethanol stress, the level of malondialdehyde (MDA)—a well-established biomarker of oxidative stress—was significantly elevated, indicating that ethanol triggered excessive ROS production and resultant oxidative damage. Given that MDA is a major product of membrane lipid peroxidation, its concentration directly reflects the integrity of the plasma membrane [54]. Correspondingly, the decrease in MDA levels observed in the Eth + Pro group (Fig. 6E) signifies an attenuation of oxidative stress and reinforces the protective role of proline in preserving membrane integrity against ethanol-induced damage (Fig. 4). Collectively, the reduction in both ROS and MDA levels upon proline supplementation demonstrates that proline alleviates ethanol stress in W. anomalus by effectively scavenging ROS and mitigating oxidative damage. This conclusion is consistent with findings in S. cerevisiae, where mutants that accumulate high endogenous proline (e.g., put1Δ, PRO1^D154N, and PRO1^I150T) similarly show reduced ROS accumulation and enhanced cell viability under ethanol stress [42].

In response to oxidative stress, yeast cells activate a sophisticated antioxidant defense system to neutralize excess ROS and maintain redox homeostasis [55]. This system primarily comprises an enzyme-dependent and a non-enzyme-dependent component. The enzyme-dependent system includes key enzymes such as superoxide dismutase (SOD) and catalase (CAT), which form a critical enzymatic cascade to mitigate oxidative damage [56]. Specifically, SOD catalyzes the conversion of superoxide radicals into hydrogen peroxide, which is then decomposed by CAT into water and oxygen, thereby preventing the formation of highly toxic hydroxyl radicals. In contrast, the non-enzyme-dependent system is a diverse network composed of endogenous molecules (such as glutathione, GSH) and exogenous nutrients (such as vitamins) [57]. It scavenges ROS through direct chemical reactions, protects biological macromolecules, and works in coordination with the enzyme-dependent system, together forming a multilayered and highly integrated defense that enables cells to cope with oxidative stress [58].

We further determined changes in this antioxidant defense system following proline addition (Fig. 6F-H). Upon exposure to ethanol stress, the activities of both SOD and CAT were significantly enhanced, accompanied by an increase in GSH content. These results indicate that both the enzyme-dependent and non-enzyme-dependent antioxidant systems were activated in response to ethanol-induced oxidative stress. Moreover, supplementation with proline led to a further enhancement in SOD and CAT activities, as well as in GSH content, which further elucidates the mechanism by which proline alleviates oxidative stress induced by ethanol.

Transcriptome analysis of W. anomalus against ethanol challenge with proline supplementation

Transcriptomics, the comprehensive study of the complete set of RNA transcripts produced by a genome, provides a powerful snapshot of gene expression under specific conditions [59]. Its key advantages include high-throughput capability, high sensitivity, and the ability to dynamically quantify expression levels, making it a frequently employed method for capturing global molecular changes in response to various stimuli [60]. By comparing transcriptome profiles between stressed and normal conditions, differentially expressed genes (DEGs), along with key regulatory networks and signaling pathways, have been investigated in yeast responses to stressors such as ethanol, furfural, acetic acid, and formic acid [22]. To elucidate the genes and their functions regulated by proline in W. anomalus under ethanol stress, a transcriptomics analysis was conducted.

RNA-seq generated a total of 43,065,443, 46,447,271, and 4,990,653 raw reads for the Control, Ethanol, and Eth + Pro groups, respectively. After quality filtering, an average of 42,245,314, 44,885,465, and 49,159,673 clean reads were retained per group. All sequencing libraries demonstrated high quality, with both Q20 and Q30 scores exceeding 90% and a GC content around 38% (Table 1). Principal component analysis (PCA) showed clear segregation among the treatment groups with high intra-group reproducibility, confirming the reliability of the samples (Fig. 7A).

Table 1.

Quality analysis of transcriptome sequencing data for each group

Groups	Raw reads	Raw bases	Clean reads	Clean bases	Error rate (%)	Q20(%)	Q30(%)	GC content (%)
Control	43,065,443	6,502,881,943	42,245,314	6,315,356,769	0.02	98.25	94.71	37.133
Ethanol	46,447,271	7,013,537,971	44,885,465	6,677,724,185	0.02	98.24	94.77	40.023
Eth + Pro	4,990,653	7,535,884,923	49,159,673	7,368,982,118	0.02	98.28	94.77	38.65

Fig. 7.

Transcriptome analysis of W. anomalus under ethanol stress with proline supplementation. A Principal component analysis (PCA) of all samples. B Volcano plot displaying differentially expressed genes (DEGs) between the Ethanol and Control groups. C Volcano plot of DEGs between the Eth + Pro and Ethanol groups. D GO enrichment analysis of DEGs from the comparison in (B). E GO enrichment analysis of DEGs from the comparison in (C). F Top 20 enriched KEGG pathways for DEGs from the comparison in (B). G Top 20 enriched KEGG pathways for DEGs from the comparison in (C)

Differential expression analysis (criteria: P < 0.05 and |log₂FC| > 2) identified 3,064 significant DEGs after 6 h of ethanol stress alone, comprising 1,568 upregulated and 1,496 downregulated genes (Fig. 7B). The addition of exogenous proline under ethanol stress modulated this response, resulting in 762 DEGs (419 upregulated, 343 downregulated) (Fig. 7C).

GO and KEGG enrichment analyses were performed to investigate the functional implications of the identified DEGs. The analysis revealed that under ethanol stress, DEGs were primarily associated with GO terms including “intracellular non-membrane-bounded organelle”, “organonitrogen compound biosynthetic process”, “oxidoreductase activity”, and “ribonucleoprotein complex” (Fig. 7D). Importantly, with proline supplementation, the most significantly enriched terms shifted to “cellular biosynthetic process”, “organic substance biosynthetic process”, “biosynthetic process”, and “cellular macromolecule metabolic process” (Fig. 7E). This shift in enrichment profiles indicates that these distinct biological processes and cellular components are likely key to W. anomalus’s adaptation to ethanol stress and to the protective regulatory effects conferred by proline.

KEGG pathway enrichment analysis was subsequently performed to identify the metabolic pathways associated with these DEGs. In the Ethanol versus Control comparison, significantly enriched pathways included ribosome, oxidative phosphorylation, peroxisome, pyruvate metabolism, and arginine and proline metabolism (Fig. 7F). Notably, following proline supplementation, the majority of DEGs were associated with pathways such as ribosome, protein processing in the endoplasmic reticulum, and ubiquitin-mediated proteolysis (Fig. 7G).

The underlying mechanism of the ethanol stress response in S. cerevisiae, a well-established model organism, has been extensively studied and is relatively well understood [17]. In contrast, the response mechanism of W. anomalus to ethanol stress remains scarcely investigated. Here, we report the pleiotropic effects of ethanol stress on W. anomalus, encompassing energy metabolism, protein biosynthesis, and nucleic acid metabolism—responses that are consistent with those observed in S. cerevisiae. Nevertheless, differences in the ethanol stress response mechanisms between non-Saccharomyces yeasts and S. cerevisiae were also identified. For example, in ethanol-stressed K. marxianus, the expression of certain genes encoding enzymes involved in unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure [61]. Conversely, these genes are upregulated in ethanol-stressed S. cerevisiae, suggesting that membrane composition restructuring differs between these two yeasts.

To ameliorate ethanol-induced damage and enhance yeast tolerance, the supplementation of exogenous nutrients and protective compounds during brewing has been widely studied and is generally accepted [13]. For instance, Chen et al. [62] investigated the protective effect of triterpenoids from Cyclocarya paliurus on S. cerevisiae under ethanol stress using transcriptomic analysis. They found that triterpenoid addition reduced filamentous and invasive growth while benefiting redox balance and glycolysis. In contrast, our present study on W. anomalus identified that the ribosomal pathway contained the highest number of DEGs following proline addition. Most of these ribosomal DEGs were upregulated in the Ethanol group but became downregulated in the Ethanol+Proline group, indicating that proline may reverse the enhanced protein synthesis triggered by ethanol stress.

The mitogen-activated protein kinase (MAPK) signaling pathways serve as central hubs in the yeast stress response network, enabling cells to sense, transduce, and adapt to a wide array of environmental challenges [63]. In this study, 16 DEGs were annotated to the MAPK signaling pathway (Supplementary Table 1). Notably, most of the MAPK signaling pathway-related DEGs were upregulated in the Eth + Pro group compared to the Ethanol group, suggesting that proline supplementation may activate MAPK pathway activity to cope with ethanol stress.

To quantitatively validate the transcriptomics results, the expression of six genes—each associated with a distinct metabolic or signaling pathway in W. anomalus—was examined by RT‑qPCR. These genes participate in ribosome biogenesis (Supplementary Fig. 2A), protein processing in the endoplasmic reticulum (Supplementary Fig. 2B), the MAPK signaling pathway (Supplementary Fig. 2C), oxidative phosphorylation (Supplementary Fig. 2D), glutathione metabolism (Supplementary Fig. 2E), and arginine and proline metabolism (Supplementary Fig. 2F). As shown in Supplementary Fig. 1, the expression patterns of the six genes (WICANDRAFT_104775, WICANDRAFT_22747, WICANDRAFT_26146, WICANDRAFT_24233, WICANDRAFT_35738, and WICANDRAFT_89804) under ethanol stress and proline supplementation were highly consistent between the RT‑qPCR and RNA‑seq analyses, confirming the reliability of the transcriptomic data.

Metabolome analysis of W. anomalus against ethanol challenge with proline supplementation

Metabolomics, the comprehensive and systematic study of the complete set of small-molecule metabolites within a biological system under defined conditions, captures a functional snapshot of cellular physiology [64]. As the final downstream product of genomic activity, the metabolome provides a direct readout of a cell’s biochemical response to various stressors. To further elucidate the protective mechanism of proline in W. anomalus under ethanol stress, we conducted a metabolomics analysis.

Based on metabolomic sequencing results, we identified 451 differentially expressed metabolites (DEMs) in W. anomalus under ethanol stress (compared to the control), comprising 220 upregulated and 231 downregulated metabolites across positive and negative ion modes, applying the criteria of variable importance in the projection (VIP) > 1 and P < 0.05 (Fig. 8A). Following proline supplementation, 98 DEMs (29 upregulated, 71 downregulated) were detected (Fig. 8B). KEGG annotation revealed 18 compound classes between the Ethanol and Control groups, with the highest number of DEMs classified as cofactors, amino acids, and phospholipids (Fig. 8C), suggesting the regulation of these compounds under ethanol stress. In contrast, only 8 compound classes were annotated in the proline-supplemented group, among which amino acids constituted the majority (Fig. 8D). Notably, the number of annotated amino acids was higher under ethanol stress with proline than under ethanol stress alone.

Fig. 8.

Metabolome analysis of W. anomalus under ethanol stress with proline supplementation. A Volcano plot of differentially expressed metabolites (DEMs) in the Ethanol vs. Control comparison. B Volcano plot of DEMs in the Eth + Pro vs. ethanol comparison. C KEGG compound classification of DEMs in (A). D KEGG compound classification of DEMs in (B). E KEGG pathway enrichment analysis of DEMs in (A). F KEGG pathway enrichment analysis of DEMs in (B)

KEGG pathway enrichment analysis showed that under ethanol stress, DEMs were primarily enriched in pathways related to cofactor biosynthesis, purine metabolism, nucleotide metabolism, ABC transporters, lysine degradation, and arginine and proline metabolism (Fig. 8E). Further analysis of the regulated cofactors revealed a decrease in oxidized glutathione (GSSG), indicating a concomitant increase in reduced glutathione (GSH), which was consistent with the colorimetric assay results (Fig. 6H). In addition, an increase in intracellular NAD⁺ (nicotinamide adenine dinucleotide) levels was detected in ethanol-treated W. anomalus. This stress-induced elevation of NAD⁺ is a fundamental metabolic switch that empowers cells to maintain vitality, execute repair, and build tolerance, positioning the NAD⁺-sirtuin axis as central to cellular resilience [65].

However, after proline addition, the enrichment profile shifted, with DEMs predominantly mapped to ABC transporters, arginine and proline metabolism, aminoacyl-tRNA biosynthesis, and D-amino acid metabolism (Fig. 8F). Pathways for lysine degradation, alanine, aspartate, and glutamate metabolism, and the biosynthesis of various secondary metabolites were also notably enriched. Notably, the “Biosynthesis of cofactors” pathway, which was significantly enriched in the Ethanol group, was not annotated following proline supplementation. Specifically, trehalose—a metabolite annotated within this pathway and a known protectant against various stresses—was significantly induced under ethanol stress. And our previous studies have proved that exogenous trehalose application promotes the survival of this W. anomalus [66, 67]. However, there were no differences in the contents of trehalose detected between the Eth + Pro group and the Ethanol group. As the compatibility solute, this phenomenon might be attributed to the replacement effect of proline on trehalose under ethanol stress conditions. Moreover, the enrichment counts for pathways related to ABC transporters, arginine and proline metabolism, and aminoacyl-tRNA biosynthesis were higher in the Eth + Pro vs. Ethanol comparison than in the Ethanol vs. Control comparison, indicating that proline likely modulates these specific pathways in response to ethanol stress in W. anomalus.

Integrated transcriptomics and metabolomics analysis of W. anomalus against ethanol challenge with proline supplementation

An integrative analysis was performed to further correlate the DEGs and DEMs of W. anomalus under ethanol challenge with proline supplementation. The results are shown in Fig. 9. Most of the significantly enriched DEGs and DEMs were associated with histidine metabolism, arginine and proline metabolism, tryptophan metabolism, and glycerophospholipid metabolism

Fig. 9.

Integrative KEGG enrichment analysis of co-expressed DEMs and DEGs

Conclusion

This study demonstrates that proline exerts a protective effect on W. anomalus during logarithmic growth under ethanol stress. Following proline supplementation, yeast cell viability as well as cellular proline transport and catabolism were enhanced. The presence of proline also mitigated ethanol-induced damage to cell morphology and ultrastructure, which was attributed to improved integrity of the cell wall, cell membrane, and mitochondria, ameliorated oxidative stress, and restored cellular homeostasis. Furthermore, proline helped maintain cell membrane homeostasis by reducing membrane permeability, increasing membrane fluidity, and elevating intracellular K⁺ levels. The alleviation of oxidative stress was associated with enhanced antioxidant enzyme activities and increased glutathione levels. Transcriptomic and metabolomic analyses indicated that proline regulated multiple biological processes, including ribosome synthesis, the cell wall integrity (CWI) pathway, ABC transporters, arginine and proline metabolism, and aminoacyl-tRNA biosynthesis. Taken together, these findings demonstrate that proline effectively protects yeast cells from ethanol-induced damage and suggest its potential application in the wine and bioethanol fermentation industries to enhance the ethanol tolerance of W. anomalus.

Methods

Yeast strain and culture conditions

The yeast strain used in this study was W. anomalus C11, originally isolated from Rosa roxburghii Tratt fruit in our previous study, and was stored in our laboratory [68]. The cryopreserved strain was first revived by streaking onto YEPD agar plates (containing 1% yeast extract, 2% peptone, 2% glucose, and 2% agar) and incubating at 28 °C for 72 h. A single colony was then inoculated into YEPD broth (1% yeast extract, 2% peptone, 2% glucose) and cultured at 28 °C with shaking at 160 rpm for 8 h to obtain log-phase cells. These cells were divided into three experimental groups: (1) the Control group, cultured in YEPD broth; (2) the Ethanol group, stressed with 9% (v/v) ethanol in YEPD; and (3) the Eth + Pro group, treated with 9% (v/v) ethanol and 50 mmol/L L-proline in YEPD. All groups were then incubated under identical conditions (28 °C, 160 rpm) for 6 h. After treatment, cells were harvested for subsequent analyses, except for the lyticase sensitivity assay. A proline-only group (without ethanol) was not included, because our experimental design focused specifically on comparing ethanol-stressed cells with or without proline supplementation, with the primary goal of evaluating its protective effect under stress conditions.

Cell viability assessment

Cell viability was first determined by the spot assay. Cells from each group were harvested by centrifugation (4,000 × g, 5 min), washed twice with distilled water, and resuspended to an OD₆₀₀ of 1.0. The suspensions were serially diluted (10⁰ to 10⁻⁴), spotted onto YEPD agar plates, and incubated at 28 °C for 36 h before being photographed under a digital microscope (Olympus, Tokyo, Japan). Cell death was monitored using methylene blue staining, and the death rate was calculated based on the number of stained (dead) cells counted across ten random fields per sample. Biomass was measured as cell dry weight, following a previously described method [22].

Scanning and transmission electron microscopy

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were carried out as previously described [22]. Briefly, cells were harvested by centrifugation (4,000 × g, 10 min), washed three times with 0.85% NaCl, and fixed in 2.5% glutaraldehyde at 4 ℃ overnight. After fixation, samples were rinsed three times with 0.1 M phosphate‑buffered saline (PBS, pH 7.4) and subsequently dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%). For SEM, dehydrated samples were critical‑point dried, sputter‑coated with gold, and observed using an FEI Quanta FEG 450 SEM (FEI, USA). For TEM, samples were embedded in epoxy resin, sectioned into 60–80 nm ultrathin slices, stained with uranyl acetate and lead citrate, and examined under an FEI Tecnai Spirit TEM operated at 120 kV.

Quantification of β-1,3-glucan and chitin

The contents of β-1,3-glucan and chitin in yeast cell walls were determined following established methods [14, 15]. For β-1,3-glucan detection, yeast cells were harvested by centrifugation (4,000 × g, 5 min), washed, and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The cells were resuspended in NaOH solution at a final concentration of 1 M and incubated at 80 °C for 30 min. Subsequently, 1.05 mL of aniline blue solution (0.18 M HCl, 0.03% aniline blue, 0.49 M glycine, pH 9.5) was added. After vortexing, the mixture was incubated at 50 °C for 30 min and then at 30 °C for an additional 30 min. Fluorescence was measured (excitation 400 nm, emission 460 nm) using a Hitachi F-4700 multimode microplate reader. Results were calculated and expressed as a percentage relative to the control.

For chitin determination, cells were harvested, washed three times with PBS, and resuspended. Calcofluor white stain was added to a final concentration of 20 µg/mL, followed by incubation at 30 °C for 5 min in the dark. After staining, cells were washed twice with PBS and resuspended in 1 mL PBS. Fluorescence intensity was recorded at 320 nm (excitation) and 430 nm (emission).

Yeast cell wall susceptibility to lyticase

The susceptibility of yeast cell walls to enzymatic digestion was evaluated based on lyticase‑induced lysis. Harvested cells were resuspended in TE buffer (10 mM Tris‑HCl, 1 mM EDTA, pH 8.0). Lyticase was added to a final concentration of 100 µg/mL, and the decrease in optical density at 600 nm (OD₆₀₀) was monitored over 70 min using a Hitachi F‑4700 multimode microplate reader (Tokyo, Japan). Measurements were recorded at 10‑min intervals to quantify the kinetics of cell wall digestion.

Cell wall integrity assay

Cell wall integrity was assessed by quantifying the release of intracellular proteins into the extracellular medium, which serves as an indicator of cell lysis and membrane damage [14, 15]. Following treatment, cultures were centrifuged (4,000 × g, 10 min), and the supernatant was collected. The amount of cytosolic protein released was then determined using a BCA assay kit (Beyotime, Shanghai, China) in accordance with the manufacturer’s protocol.

Assessment of cell membrane integrity, permeability, and fluidity

The integrity of the cell membrane was evaluated by dual staining with fluorescein diacetate (FDA) and propidium iodide (PI) [14, 15]. Cells were incubated with 20 µM FDA or PI at 25 °C for 25 min with shaking at 150 rpm, then visualized using an Olympus BX51 fluorescence microscope (Tokyo, Japan) and quantified with a Hitachi F‑4700 fluorescence spectrophotometer (Tokyo, Japan). Membrane permeability was assessed using a relative electrical conductivity assay. Membrane fluidity was determined by measuring the generalized polarization (GP) of the fluorescent probe Laurdan, following the method described by Jin et al. [35].

Determination of relative proline content

Intracellular proline content was measured using a proline assay kit (Jiancheng, China) according to the manufacturer’s instructions. Relative proline content was calculated by normalizing the values of the ethanol‑treated and ethanol + proline‑treated groups against that of the control group.

Determination of intracellular K⁺ concentration

Intracellular K⁺ concentration was measured using a commercial assay kit (C001‑2‑1; Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s instructions. Briefly, yeast cells were harvested by centrifugation at 4,000×g for 5 min, washed twice with PBS (pH 7.4), and resuspended in 1 mL of PBS. The cell suspension was then sonicated on ice using an ultrasonic disruptor at 150 W with alternating 5‑s on/off cycles for a total of 4 min. The resulting lysate was centrifuged at 12,000×g for 10 min at 4 °C, and the supernatant was collected for subsequent measurements of K⁺ concentration.

Determination of mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨm) was assessed qualitatively and quantitatively using the fluorescent probe rhodamine 123 (C2007; Beyotime Biotech, China). For qualitative analysis, fluorescence images were captured using a fluorescence microscope (Olympus BX51, Japan). For quantitative measurement, fluorescence intensity was recorded using a fluorescence spectrophotometer (Hitachi F‑4700, Japan).

Determination of ATP concentration

ATP concentration was measured using an ATP Assay Kit (A095‑1; Jiancheng Bioengineering Institute, China) according to the manufacturer’s protocol. Cell samples were prepared following the same procedure as described for the mtETC complex activity assay: cells were collected by centrifugation, disrupted by ultrasonication, and the resulting lysate was centrifuged to obtain the supernatant. The supernatant was then mixed with the detection reagent and incubated at 37 °C for 10 min. Absorbance was recorded at 636 nm, and ATP content was calculated using the formula supplied in the kit instructions.

Determination of intracellular ROS levels

Intracellular ROS levels were determined by measuring hydrogen peroxide and superoxide anion using the fluorescent probes DCFH‑DA (S0034S; Beyotime Biotech, China) and DHE (S0064S; Beyotime Biotech), respectively. Qualitative assessment was performed using fluorescence microscopy (Olympus BX51, Japan), while quantitative analysis was conducted using a fluorescence spectrophotometer (Hitachi F‑4700, Japan). Both probes were used at working concentrations of 10 µM (DCFH‑DA) and 4 µM (DHE), following the manufacturer’s protocols.

Determination of SOD and CAT activities

The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) were determined using the Total SOD Assay Kit (A001‑1; Jiancheng Bioengineering, China) and the CAT Assay Kit (A007‑1‑1; Jiancheng Bioengineering), respectively, following the manufacturers’ protocols. Briefly, W. anomalus cells were harvested by centrifugation at 6,000 rpm for 10 min, washed twice with phosphate‑buffered saline (PBS, pH 7.4), and resuspended in 1 mL of PBS. The suspensions were sonicated using an ultrasonic cell disruptor at 150 W with alternating 5‑s on/off cycles for a total of 4 min, followed by centrifugation at 12,000 rpm for 10 min. The resulting supernatant was collected for enzyme activity assays. Subsequently, 200 µL of substrate working solution was added, and the mixture was incubated at 37 °C for 20 min. Absorbance was measured at 450 nm (for SOD) and 405 nm (for CAT) using a microplate reader. One unit of SOD activity was defined as the amount of enzyme required for 50% inhibition, whereas one unit of CAT activity was defined as the amount decomposing 1 µmol of H₂O₂ per minute per milligram of protein.

Determination of glutathione and malondialdehyde concentrations

Intracellular concentrations of reduced glutathione (GSH) and malondialdehyde (MDA) were measured using a GSH and GSSG Assay Kit (S0053; Beyotime Biotech, China) and an MDA Assay Kit (A003‑3; Jiancheng Bioengineering, China), respectively, following the manufacturers’ protocols. Briefly, cells were harvested by centrifugation at 6,000 rpm for 10 min, washed twice with PBS (pH 7.4), and resuspended in PBS. The suspensions were sonicated using an ultrasonic cell disruptor at 150 W with alternating 5‑s on/off cycles for 4 min, and then centrifuged at 12,000 rpm for 10 min. The resulting supernatant was collected for subsequent assays.

For GSH measurement, the supernatant was used directly. Then, 150 µL of total glutathione detection working solution and 50 µL of NADPH (0.5 mg/mL) were added, and the mixture was incubated at 25 °C for 5 min. Absorbance was recorded at 412 nm, and total GSH was calculated using a standard curve. For MDA determination, the supernatant was processed according to the kit instructions, and absorbance was measured at 530 nm. MDA concentration was determined against a standard curve.

Transcriptomics analysis

Total RNA from Control, Ethanol, and Ethanol + Pro group cells was isolated with TRIzol reagent (Invitrogen, USA) per the manufacturer’s instructions, followed by DNase I (Takara, Japan) treatment to remove genomic DNA. Using 1 µg of this RNA, a transcriptome library was prepared with the TruSeqTM RNA Sample Preparation Kit (Illumina, USA). The library construction involved size-selecting ~ 300 bp cDNA fragments on a 2% Low Range Ultra Agarose gel and amplifying them via 15-cycle PCR with Phusion DNA polymerase (NEB, USA). Finally, paired-end sequencing was performed on an Illumina HiSeq X Ten/NovaSeq 6000 platform. Differentially expressed genes (DEGs) were defined by |fold change| > 2 and P-value < 0.05, and subsequently analyzed for KEGG pathway enrichment on the Majorbio Cloud Platform (https://cloud.majorbio.com), adhering to its protocol and the method of Li et al. [22].

Metabolomics analysis

Non-targeted metabolomics was performed by Majorbio Biotech Co., Ltd. (Shanghai, China) on a Vanquish Horizon liquid chromatography–mass spectrometry system (Thermo Scientific, Waltham, MA, USA). Briefly, 50 mg of cells from the Control, Ethanol, and Eth + Pro groups were homogenized in 400 µL of ice-cold methanol/water (4:1, v/v) containing 0.02 mg/mL L-2-chlorophenylalanine as an internal standard. The mixture was vortexed thoroughly for 30 s and subsequently sonicated at 40 kHz for 30 min at 5 °C to maximize metabolite extraction. After incubation at − 20 °C, samples were centrifuged at 13,000 × g for 15 min, and the resulting supernatant was collected for LC-MS analysis. Chromatographic and mass spectrometric parameters followed the method established by Li et al. [22]. Raw LC-MS data were processed with Progenesis QI 2.3 software (Waters, Milford, MA, USA) for peak picking, alignment, and data matrix construction, containing retention time, m/z, and intensity information. Differentially expressed metabolites (DEMs) were screened using criteria of variable importance in projection (VIP) > 1.0 and a P-value < 0.05. These DEMs were then annotated and functionally interpreted through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the Majorbio Cloud Platform, conducted in accordance with the platform’s operational guidelines.

Real-time quantitative PCR (RT-qPCR) analysis

Total RNA was isolated from W. anomalus cells and purified using a commercial kit (Takara, Japan), and reverse-transcribed into cDNA with the PrimeScript RT Reagent Kit (Takara, Japan), following the manufacturer’s protocols. RT-qPCR was performed on a LightCycler 96 system (Roche, Germany) using the primers listed in Supplementary Table 2. The thermal cycling conditions were as follows: initial denaturation at 95 ℃ for 30 s; 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. A melting curve analysis was conducted from 95 ℃ to 97 ℃ to confirm amplification specificity. Gene expression levels were normalized to ACT1 and calculated via the 2^−ΔΔCT method.

Statistical analysis

Data are expressed as the mean ± SD from three biologically independent replicates. Prior to analysis, all statistical evaluations were performed with SPSS 21.0 software. Inter-group differences were determined by one-way ANOVA coupled with Duncan’s post hoc test, where a P-value less than 0.05 indicated statistical significance. For graphical representation, column charts were created using GraphPad Prism 10.1.2 (GraphPad Software, USA).

Authors’ contributions

Xiaozhu Liu: Conceptualization, Data curation, Funding acquisition, Methodology, Writing – Original draft. Yujie Wang: Data curation, Investigation, Methodology, Software. Guilan Jiang: Data curation, Investigation. Shuangyan Liu: Data curation, Investigation. Yinfeng Li: Data curation, Funding acquisition, Project administration, Writing – Review & editing.

Funding

This study was supported by the National Natural Science Foundation of China(32160557); Guizhou Provincial Science and Technology Foundation (Qiankehejichu MS [2026]244; [2025]192;[2025] 196).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The transcriptomics datasets presented in this study are available online at NCBI ( https://www.ncbi.nlm.nih.gov/ ) under accession number PRJNA1468413.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.