Abstract Sugarcane molasses is an ideal economical raw material for ethanol production because of its wide availability, low cost and nutrient content. However, benzoic acid compounds with toxic effects on yeast cells are commonly found in sugarcane molasses. At present, the molecular mechanism of the toxic effects of benzoic acid on Saccharomyces cerevisiae has not been elucidated. Here, the toxic effect of exogenous benzoic acid on S. cerevisiae GJ2008 cells was studied, and the genes differentially expressed in S. cerevisiae GJ2008 after 1.2 g/L benzoic acid stress were identified via Illumina RNA-Seq technology. The results indicated that benzoic acid significantly inhibited yeast cell growth, prolonged their rapid growth period, and ultimately reduced their biomass. During ethanol fermentation using 250 g/L sucrose under 1.2 g/L benzoic acid stress, several adverse effects were observed, such as high residual sugar content, low ethanol concentration and low fermentation efficiency. In addition, the cell morphology was damaged, the cell membrane permeability increased, intracellular nucleic acid and protein leakage increased, and the malondialdehyde content significantly increased. Moreover, the cells protected themselves by significantly increasing the intracellular glycerol content. Fourier transform infrared spectroscopy proved that benzoic acid could reduce the degree of unsaturation and increase cell membrane permeability by changing the yeast cell wall and cell membrane composition, leading to cell damage and even death. Transcriptomic analysis revealed that under benzoic acid stress, the expression of genes associated with sucrose and starch metabolism, thiamine metabolism, the glycolysis pathway, fructose and mannose metabolism, galactose metabolism and ABC transporters was significantly downregulated. The expression of genes related to ribosomes, lipid metabolism, ribosome biosynthesis, nucleic acid metabolism, arginine and proline metabolism, RNA polymerase, metabolism related to cofactor synthesis, and biosynthesis of valine, leucine, and isoleucine was significantly upregulated. These results indicated that benzoic acid inhibited glycolysis and reduced sugar absorption and utilization and ATP energy supply in yeast cells. In response to stress, genes related to the ribosome bioanabolic pathway were upregulated to promote protein synthesis. On the other hand, the expression of ELO1, SUR4, FEN1 and ERG1 was upregulated, which led to extension of long-chain fatty acids and accumulation of ergosterol to maintain cell membrane structure. In conclusion, this paper provides important insights into the mechanism underlying the toxicity of benzoic acid to yeast cells and for realizing high-concentration ethanol production by sugarcane molasses fermentation. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-80484-1. Keywords: Benzoic acid, Saccharomyces cerevisiae, Ethanol fermentation, Physicochemical properties, Transcriptomic Subject terms: Biological techniques, Biotechnology Introduction Sugarcane molasses is an ideal economical raw material for ethanol production because of its wide availability and low cost^[28]1. It contains nutrients such as carbon sources, nitrogen sources, inorganic salts and vitamins necessary for yeast growth^[29]2; it is mainly used for fermentation to produce ethanol and can be processed into alcohol for consumption, for use in medicine and for use as fuel^[30]1. At present, the ethanol yield from fermentation using sugarcane molasses in factories is confronted with problems such as low alcohol yield, high energy consumption and low production efficiency^[31]3. It is difficult to achieve high-concentration ethanol production via fermentation, which severely restricts the efficient utilization of molasses. This is mainly due to the organic acids, phenolic compounds, metal ions, colloidal substances, caramel pigments and other components contained in molasses, which are closely related to the inhibition of yeast cell growth and metabolism^[32]4. Payet et al.^[33]5 tested the components of sugarcane molasses from different regions using LC‒MS technology and detected p-coumaric acid, ferulic acid and benzoic acid. Phenolic compounds in sugarcane molasses are mainly biosynthesized from pentose phosphate, shikimic acid and phenylpropyl during sugarcane growth, and in the sugar production process, the phenolic substances combine with iron ions to form dark complex phenolic pigments^[34]6,[35]7. Larsson et al.^[36]8 suggested that the toxic effect of phenolic compounds on cells is related to their relative molecular weight, wherein the lower the molecular weight is, the greater the toxicity. The relative molecular weight of benzoic acid (C[7]H[6]O[2]) is lower than that of coumaric acid (C[9]H[8]O[3]) and ferulic acid (C[10]H[10]O[4]), and it is speculated that its toxic effect is stronger than that of other phenols in sugarcane molasses^[37]9,[38]10. Saccharomyces cerevisiae is the main microorganism used in ethanol fermentation of molasses. There are few reports on the mechanism underlying the toxicity of phenolic substances in sugarcane molasses to S. cerevisiae. Therefore, in this study, the effect of benzoic acid on the ethanol fermentation process and physicochemical parameters of S. cerevisiae was explored. In recent years, transcriptomic methods have been used to explore the mechanism underlying the microbial response to harmful stress. Guo et al.^[39]11 explored the genetic mechanism of nucleic acid synthesis in the mutant strain S. cerevisiae BY23-195, which has a high nucleic acid content, through transcriptome sequencing technology and revealed that the RNA content of S. cerevisiae could be increased by regulating the transcription of HXT1, GSP2 and CTT1. Zeng et al.^[40]12 studied the response and tolerance mechanism of S. cerevisiae to formic acid stress and found that the expression of most genes related to glycolysis, glycogen synthesis, protein degradation, the cell cycle, the MAPK signaling pathway and reoxidation regulation was significantly induced under formic acid stress, and that of amino acid synthesis-related genes involved in protein translation and synthesis was significantly inhibited. Energy metabolism may be important in the adaptation of S. cerevisiae to formic acid. Li et al.^[41]13 reported that under acetic acid and furfural stress conditions, the overexpression of Haa1p or Tye7p increased the xylose consumption rate by nearly 50% and the ethanol yield by nearly 8%. Jin et al.^[42]14 performed a transcriptomic analysis of S. cerevisiae under 2-phenylethanol stress and reported that 2-phenylethanol inhibited the synthesis of plasma membrane proteins, thereby inhibiting the transport of nutrients required for growth. Ismail et al.^[43]15 showed that adding metal ions to the growth medium under acetic acid stress could increase the tolerance of S. cerevisiae to acetic acid. Bereketoglu et al.^[44]4 reported that exposure of Saccharomyces cerevisiae to 300 mg/L BPA led to severe changes in the expression levels of several genes involved in oxidative phosphorylation, the tricarboxylic acid cycle, ribosome activity, replication and chemical reactions. The response mechanism of S. cerevisiae to pest stress was studied by means of omics sequencing, which laid a foundation for identifying key genes and applying metabolic engineering for strain modification. To further study the mechanism underlying the regulatory effect of benzoic acid on yeast metabolism during ethanol fermentation using sugarcane molasses, transcriptomics was used. In this paper, the highly sugar-tolerant S. cerevisiae GJ2008 strain was used, and benzoic acid was added to the fermentation medium supplemented with 250 g/L sucrose to analyze its effects on sucrose hydrolysis, fructose and glucose consumption, ethanol production and the overall performance of S. cerevisiae in ethanol fermentation. Moreover, the effects of benzoic acid on the growth of S. cerevisiae cells, cell membrane permeability, malondialdehyde (MDA) content and intracellular glycerol content were determined. The morphology of the yeast cells was observed by scanning electron microscopy (SEM), and the changes in the cell wall and cell membrane structure were analyzed by Fourier transform infrared (FTIR) spectroscopy. In addition, RNA-Seq technology was used to analyze the response mechanism of S. cerevisiae cells under benzoic acid stress. Materials and methods Yeast strain and culture conditions The strain S. cerevisiae GJ2008 used in this study is an industrial alcohol fermentation yeast strain with a high tolerance to sucrose and was provided by the Institute of Fermentation Engineering, Guangxi University of Science and Technology. S. cerevisiae GJ2008 was precultured in YPD medium (2% glucose, 2% peptone, 1% yeast extract) at 30 °C and 150 rpm for 12 h; after two rounds of activation, the GJ2008 yeast cells were inoculated into a fermentative medium (25% sucrose, 2% peptone, 1% yeast extract). To determine the effect of benzoic acid on the growth of strain GJ2008, an appropriate amount of benzoic acid was added to the fermentative medium to produce final concentrations of 0.5, 1.0, 1.2, 1.4, 1.6 and 1.8 g/L (0.5% DMSO was used as a cosolvent), and the solution without benzoic acid was used as a control group. Strain GJ2008 was cultured with agitation at 150 rpm at 30 °C in shake flasks, and samples were taken every 2 h for determination of the optical density (OD) at a wavelength of 560 nm. Based on the growth state of strain GJ2008 under different benzoic acid concentrations, benzoic acid was used at a concentration of 1.2 g/L for subsequent experiments. Analysis of the ethanol fermentation process 1.2 g/L benzoic acid was added to the fermentative medium, and medium without benzoic acid was used as a control. The activated yeast cells were inoculated in a 500 mL flask containing 200 mL medium and then cultured at 30 °C and 150 rpm for 36 h. Samples were taken every 3 h and centrifuged at 10,000 rpm for 10 min at 4 °C. The cell pellets were resuspended for OD measurements at 560 nm using a UV‒vis spectrophotometer, and the supernatants were filtered through a 0.22 μm filter membrane before determination of the concentrations of sucrose, glucose, fructose and ethanol by high-performance liquid chromatography (HPLC, Agilent 1260, USA). The HPLC conditions used were as follows: an Alltima 5 μm Amino (Agilent, 250 × 4.6 mm) column with an acetonitrile-water (75:25, v/v) mobile phase, a refractive index detector (RID), and a flow rate of 1 mL/min. Gas chromatography (GC, Agilent 8890, USA) was used to determine the ethanol content, and the conditions used were as follows: a TM-930 25 m×0.53 mm×1 μm column with a flame ionization detector (FID); the initial temperature of the column was 40 °C, which was increased to 80 °C at a rate of 5 °C/min for 2 min and then increased to 150 °C at a rate of 10 °C/min; the inlet temperature was 180 °C; the injection volume was 0.5 µL; and the split ratio was 20:1. Cell morphology analysis The yeast cells were cultured in fermentative medium to the exponential growth stage, treated with 1.2 g/L benzoic acid for 3 h, and then collected by centrifugation at 4 °C. The cell pellets were resuspended and washed three times in PBS. Glutaraldehyde (2.5%) was used for immobilization, and the samples were then eluted with different ethanol concentrations^[45]16. After freeze-drying, the yeast cells were observed by SEM (Quattro S FE-SEM, Japan). Cell membrane permeability analysis Cell membrane permeability was evaluated by measuring the leakage of nucleic acids and proteins in cells according to the methods of Xiang et al.^[46]17 Yeast cells were cultured in fermentative medium to the exponential growth stage and then treated with 1.2 g/L benzoic acid. Samples treated for 0 h, 3–6 h were collected. The enzymes in the fermentation broth were inactivated by placing the samples in a water bath at 80 °C for 5 min, and the samples were then centrifuged at 6000 rpm for 10 min. The absorbance of nucleic acids and proteins in the supernatant was measured at 260 nm and 280 nm, respectively. Malondialdehyde content analysis Yeast cells treated with 1.2 g/L benzoic acid for 0 h, 3–6 h were collected, washed and resuspended, after which 10% trichloroacetic acid (w/v) extract and 0.6% thiobarbituric acid (TBA) reagent were added and fully mixed^[47]18. The samples were boiled in a water bath at 100 °C for 15 min and centrifuged at 8000 rpm for 5 min after rapid cooling. The absorbance of the supernatant at 450 nm, 532 nm and 600 nm was measured, with 0.6% TBA used as a blank control. The cell dry weight was measured, and the malondialdehyde (MDA) concentration was calculated^[48]19. Analysis of the intracellular glycerol content Yeast cells treated with 1.2 g/L benzoic acid for 6–12 h were collected, and ultrasonic fragmentation was conducted at low temperature. The supernatant was collected by centrifugation at 12,000 rpm and 4 °C for 10 min, and the glycerol content was determined according to the instructions of the glycerol content determination kit. FTIR analysis Yeast cells treated with 1.2 g/L benzoic acid for 3 h were collected and freeze-dried. The freeze-dried yeast and potassium bromide were fully ground, mixed evenly, kept dry and tableted. The infrared adsorption spectra were measured by FTIR spectroscopy. Transcriptome and qRT‒PCR analysis Yeast cells were cultured in fermentative medium to the exponential growth stage and then treated with 1.2 g/L benzoic acid for 2 h; cells that were not treated with benzoic acid were used as the control group. The yeast cells were harvested by centrifugation at 8000 rpm for 3 min at 4 °C, and after three washes, the cells were immediately frozen in liquid nitrogen to stop cellular metabolism. Total RNA was extracted using TRIzol reagent. Messenger RNA was first isolated by oligo(dT) beads according to the poly(A) selection method and then fragmented by fragmentation buffer. Second, double-stranded cDNA was synthesized using a SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) with random hexamer primers (Illumina). Then, the synthesized cDNA was subjected to end repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. PCR amplification was performed to obtain the sequencing library, which was subsequently sequenced on an Illumina NovaSeq 6000 sequencer by Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China). After the quality assessment of the sequencing results, the RSEM (RNASeq by Expectation Maximization) tool was used to calculate the gene expression level, and the DESeq2 algorithm was used to screen the differentially expressed genes (DEGs) between the samples with the fold change (|log[2]FC|≥1) and the significant level (p-adjusted < 0.05) as the threshold. Gene description and annotation were performed in the Genome Database of Saccharomyces cerevisiae ([49]https://www.ncbi.nlm.nih.gov/genome). GO enrichment and KEGG pathway enrichment analysis of DEGs were performed using DAVID database ([50]https://david.ncifcrf.gov/) and KOBAS database ([51]http://kobas.cbi.pku.edu.cn/)^[52]20–[53]22. To verify the reliability of the transcriptome sequencing data, 9 key genes were selected for qRT‒PCR analysis. The qRT‒PCR samples were prepared using SYBR Green PCR Master Mix (Promega, USA), and the primers used are listed in Table [54]S1. The amplification and analysis were performed according to Zeng et al.^[55]23. Statistical analysis Significance analysis of the experimental data was performed by one-way ANOVA (GraphPad Prism 8.1 software) (p < 0.05), and the data were plotted with Origin 9.5 software. Results Effect of benzoic acid concentration on the growth of strain GJ2008 To determine the effect of benzoic acid on ethanol fermentation by S. cerevisiae, we first analyzed its effect on the growth of strain GJ2008. The growth curves of S. cerevisiae GJ2008 with the addition of different concentrations of benzoic acid are presented in Fig. [56]1. With increasing benzoic acid concentration (0–1.8 g/L), the degree of cell growth inhibition gradually increased. Fig. 1. [57]Fig. 1 [58]Open in a new tab Growth curves and growth rate of S. cerevisiae GJ2008 under different concentrations of benzoic acid. As shown in Fig. [59]1A, the control group (without benzoic acid) entered the rapid growth phase at 2 h and reached the stable phase at 8 h. The time required to enter the rapid growth phase and stable phase markedly increased as the benzoic acid concentration increased to the range of 0.5–1.4 g/L. When the concentration of benzoic acid was greater than 1.6 g/L, the growth of GJ2008 was almost completely inhibited. According to Fig. [60]1B, the OD[560] in the control group increased at a faster rate than that in the benzoic acid treatment group in the first 6 h, and the increase rate was close to zero in the control group after 8 h, indicating that the growth of the yeast cells had reached a stable stage. With increasing benzoic acid concentration, the maximum increase rate of OD[560] was delayed. According to the results, 1.2 g/L benzoic acid was selected for further study of the effects of benzoic acid on the ethanol fermentation process, physicochemical parameters and transcriptome analysis of S. cerevisiae GJ2008. Effect of benzoic acid on the ethanol fermentation process of strain GJ2008 To explore the effect of benzoic acid on ethanol fermentation by S. cerevisiae GJ2008, 250 g/L sucrose was used to simulate the fermentable sugar in sugarcane molasses for ethanol fermentation experiments. In the process of ethanol fermentation by S. cerevisiae using sucrose, the intracellular sucrose hydrolase of yeast is secreted to the extracellular space, and sucrose is hydrolyzed to produce glucose and fructose, which are used by yeast cells to grow and produce ethanol. The changes in sucrose, fructose and glucose concentrations and the associated rates during ethanol fermentation using 250 g/L sucrose under 1.2 g/L benzoic acid stress are shown in Fig. [61]2. Fig. 2. [62]Fig. 2 [63]Open in a new tab Changes in sucrose, fructose and glucose concentrations (A) and the associated rates (B) during ethanol fermentation using 250 g/L sucrose under benzoic acid stress. CK, control group; BA, benzoic acid treatment group. The results shows that the trends of sucrose hydrolysis, glucose consumption and fructose consumption were similar between the treatment group and the control group (Fig. [64]2). In the control group, sucrose hydrolysis and glucose consumption were basically complete at 21 h of fermentation, indicating that by this stage, a large amount of sucrose hydrolase had been released, and the cells could grow rapidly using glucose and fructose. In the benzoic acid treatment group, sucrose was completely hydrolyzed at 36 h of fermentation, and the utilization of glucose and fructose was lower than that in the control group. It is speculated that benzoic acid may affect the sucrose hydrolase activity and sugar utilization ability of yeast. In addition, the consumption of glucose is greater than that of fructose, which may be due to the different affinities of the hexose transmembrane transporter for glucose and fructose^[65]17. The above results indicate that the sucrose hydrolysis rate and sugar utilization rate were low under the stress of benzoic acid, which was likely due to incomplete ethanol fermentation using 250 g/L sucrose. As shown in Fig. [66]3, in the control group, there was no obvious lag phase in cell growth, and the cells could rapidly use sugar to grow and reproduce and generate a large amount of ethanol (122.69 g/L). However, in the benzoic acid treatment group, the residual sugar concentration was still as high as 97.39 g/L, and the alcohol concentration was only 75.35 g/L at 36 h of fermentation. The residual sugar content increased by 91.24%, the ethanol content decreased by 38.59%, and the OD value (5.77) was much lower than that of the control group. In general, benzoic acid inhibited the growth of yeast cells and reduced the ability of cells to hydrolyze sucrose, utilize sugars and produce ethanol. Fig. 3. [67]Fig. 3 [68]Open in a new tab Biomass, total residual sugar and ethanol production curves for ethanol fermentation using 250 g/L sucrose under benzoic acid stress. The total sugar fermentation efficiency is an important index for evaluating the effect of ethanol fermentation. When ethanol fermentation was carried out at the same sugar concentration (25%), a lower residual total sugar concentration and a higher ethanol concentration indicated a better fermentation effect. As shown in Table [69]1, compared with those of the control group, the final ethanol concentration, glucose utilization rate, fructose utilization rate, total sugar utilization rate, total sugar fermentation efficiency and sugar consumption fermentation efficiency of the benzoic acid treatment group decreased by 35.58%, 24.76%, 44.76%, 34.37%, 37.61% and 4.93%, respectively. The final ethanol concentration (75.35 g/L) and total sugar fermentation efficiency (55.15%) of the treatment group were lower, which was related to the high residual total sugar content (97.39 g/L) and low utilization rates for glucose and fructose (75.24% and 51.74%, respectively). Table 1. Results of ethanol fermentation using 250 g/L sucrose. Concentration of benzoic acid(g·L^−1) Total sugar concentration of 0 h (g·L^−1) The residual total sugar concentration (g·L^−1) The residual sucrose concentration (g·L^−1) The residual fructose concentration (g·L^−1) The residual glucose concentration (g·L^−1) Ethanol concentration (g·L^−1) The utilization of glucose (%) The utilization of fructose (%) The utilization of total sugar (%) Fermentation efficiency of total sugar (%) Fermentation efficiency of Consumed sugar (%) 0 271.62 8.54 0 8.54 0 122.69 100 93.67 96.86 88.40 91.26 1.2 267.36 97.39 1.49 63.34 32.49 75.35 75.24 51.74 63.57 55.15 86.76 [70]Open in a new tab In summary, the exogenous addition of 1.2 g/L benzoic acid significantly inhibited the growth of yeast cells, reduced cell viability, and caused yeast cells to metabolize sugar to produce ethanol, resulting in a high residual sugar content, low ethanol production and low fermentation efficiency. Effect of benzoic acid on cell morphology Under high permeability stress, strong acid or alkali stress, and inhibitor stress, the cell membrane is destroyed first^[71]24. Therefore, the effect of benzoic acid on the morphology of S. cerevisiae cells was observed by SEM. Figure [72]4 shows that the cells in the control group were oval or round in shape and were full, smooth, flat, and compact, with many bud marks and no ruptures or pores. The shape of the yeast cells in the treatment group was irregular, the surface was uneven, and the number of buds was small. A large number of voids were observed, along with intercellular adhesion. In summary, on the one hand, benzoic acid destroyed the integrity of the cell membrane and cell wall of S. cerevisiae, causing intracellular leakage and intercellular adhesion; on the other hand, it affected the budding of yeast cells, thereby affecting the normal growth and reproduction of cells. Fig. 4. [73]Fig. 4 [74]Open in a new tab Scanning electron microscopy images of S. cerevisiae GJ2008 in the control group (CK) and benzoic acid treatment group(BA).Rred circles indicating the damage on the surface of the strain. Effect of benzoic acid on membrane permeability Nucleic acids and proteins are important biological macromolecules in cells that do not leak out of cells when the cell membrane permeability is normal^[75]25. The conjugated double bonds of purine and pyrimidine bases in nucleic acids result an absorbance peak at 260 nm. The tryptophan and phenylalanine residues in proteins contain benzene rings, which result in an absorbance peak at 280 nm. Therefore, the OD[260] and OD[280]values of the fermentation supernatant determined by spectrophotometry can not only reflect the leakage of nucleic acids and proteins but also indirectly reflect the degree of cell membrane damage^[76]26,[77]27. Figure [78]5 shows that after treatment with benzoic acid, the protein and nucleic acid release from yeast cells increased significantly (p < 0.05), and the longer the treatment time was, the greater the release. Compared with those in the control group, the amount of nucleic acid and protein released increased 1.80 and 2.06 times, respectively, after 3 h of benzoic acid treatment. After 6 h of treatment, the nucleic acid and protein contents increased 2.67- and 2.31-fold, respectively. The above results indicated that under benzoic acid stress, intracellular nucleic acids and proteins leaked out of the cell, and the permeability of the yeast cell membrane increased as a result of severe damage. Fig. 5. [79]Fig. 5 [80]Open in a new tab Changes in the membrane permeability of S. cerevisiae GJ2008 under benzoic acid stress. ns indicates that there was no significant difference between the benzoic acid treatment group and the control group; * and ** indicate p < 0.05 and p < 0.01, respectively. Effect of benzoic acid on the MDA content in yeast cells MDA is produced by polyunsaturated fatty acid peroxidation catalyzed by active oxygen^[81]28,[82]29. The change in MDA content can indicate the degree of membrane lipid peroxidation. In addition, MDA can destroy protein structure and increase the toxic effect of reactive oxygen species, further changing the fatty acid composition of the cell membrane, reducing the fluidity of the cell membrane, increasing cell membrane permeability, and damaging cell membrane integrity^[83]30. MDA and TBA can produce reddish-brown trimethylamine under acidic heating conditions, and trimethylamine has an absorption peak at 532 nm. The MDA content in S. cerevisiae cells before and after benzoic acid treatment was measured and is shown in Fig. [84]6. The intracellular MDA content in yeast cells increased significantly after benzoic acid treatment (p < 0.05) and was positively correlated to the treatment time. The MDA content of the yeast cells increased 4.25- and 4.51-fold after benzoic acid stress for 3 h and 6 h, respectively, compared to that in the control group. The MDA content increased significantly at 3 h, indicating that benzoic acid is highly toxic to yeast cells and can cause severe oxidative damage in a short time. The above results showed that S. cerevisiae caused lipid peroxidation of the cell membrane under benzoic acid stress, and the MDA content increased significantly. This finding is consistent with the above results of SEM showing intracellular nucleic acid and protein leakage and changes in cell morphology. Fig. 6. Fig. 6 [85]Open in a new tab Changes in the intracellular MDA content in S. cerevisiae GJ2008 under benzoic acid stress. ns indicates that there was no significant difference between the benzoic acid treatment group and the control group; *** indicates p < 0.001. Effect of benzoic acid on the intracellular glycerol content The intracellular glycerol content is an important indicator of yeast tolerance. Many studies have shown that yeast cells increase intracellular glycerol production to balance osmotic pressure and reduce cell damage under unfavorable growth conditions^[86]31,[87]32. As shown in Fig. [88]7, there was no significant difference in the glycerol content in the control group within 12 h of benzoic acid treatment. In the treatment group, the intracellular glycerol content increased significantly with increasing treatment time (p < 0.05). At 6 h and 12 h, the intracellular glycerol content increased 1.48- and 1.50-fold, respectively, compared with that in the control group. These results indicated that benzoic acid stress induced glycerol synthesis and accumulation in cells to protect cells from damage. Fig. 7. [89]Fig. 7 [90]Open in a new tab Changes in the intracellular glycerol content in S. cerevisiae GJ2008 under benzoic acid stress. * indicate that there was a significant difference between the benzoic acid treatment group and the control group; * and ** indicate p < 0.05 and p < 0.01, respectively. FTIR spectroscopy FTIR spectroscopy was used to characterize the changes in the cell structure components of S. cerevisiae GJ2008 after 1.2 g/L benzoic acid treatment, and the results are shown in Fig. [91]8. The characteristic FTIR bands shown are basically consistent with previous research reports^[92]33–[93]35. There was a strong and wide absorption peak between 3500 and 3100 cm^−1, which are attributed to the deformation and vibration of O-H and N-H bonds in proteins, fatty acids, polysaccharides, chitin and other substances^[94]36. The absorption peaks at 3100 –2800 cm^−1 represent lipid functional groups and are attributed to the antisymmetric stretching vibration of the -CH group of fatty acids in triacylglycerol^[95]37. The amide I, II and III bands near 1650 cm^−1, 1542 cm^−1 and 1242 cm^−1 are the most important bands for protein characterization. The absorption peak of the amide I band is attributed to the stretching vibration of C = O and the bending vibration of N-H, which indicates that the protein structure in yeast cells consists mainly of α-helices. The absorption peak in the amide II zone is attributed to N-H bending vibrations and C-N stretching vibrations. The absorption peak at the amide III band is attributed to the bending vibration of the C-H bond, the stretching vibration of C-O and the deformation vibration of P = O in the carboxyl group, which indicates that it is related to the phospholipid bilayer and represents the asymmetric stretched phosphodiester bond. The characteristic peaks around 1454 cm^−1 are attributed to the shear vibration of CH[2], the asymmetric bending vibration of CH[3] and the antisymmetry of the carboxylate group RCOO-. The characteristic peak at 1080 cm^−1 is attributed to the vibration of the polysaccharide hydroxyl skeleton in the nucleic acid or cell wall. The absorption peak below 1000 cm^−1 is the characteristic absorption peak of yeast^[96]36. Fig. 8. [97]Fig. 8 [98]Open in a new tab Changes in the Fourier transform infrared spectra of Saccharomyces cerevisiae GJ2008 under benzoic acid stress. Changes in the structure or configuration of the compound leads to an energy level difference between the ground state and the excited state and to changes in the first excited state and the ground state, resulting in changes in the energy of photon absorption or emission, leading to a redshift or blueshift in the spectrum. As shown in Fig. [99]8, the absorption peaks of protein amide II, amide III, and polysaccharide hydroxyl groups at 1542, 1242, and 1080 cm^−1 shifted to 1537, 1238, and 1070 cm^−1 under benzoic acid stress, respectively, indicating that the vibration spectrum moved toward a low wavenumber and that a redshift occurred, the energy required for vibration decreased, and the groups became more unstable. The characteristic peak at 3355 cm^−1 shifted to 3424.5 cm^−1, and a blueshift occurred, indicating that benzoic acid affected the lipid composition of the yeast cell membrane. In summary, benzoic acid affects the structure of phospholipid fatty acids, proteins and the cell wall polysaccharide chitin on the cell membrane, increases the permeability of the cell membrane, causes the leakage of intracellular nucleic acids and proteins, and inhibits the growth and metabolism of cells. This result is consistent with the results of physical and chemical parameter measurements and similar to the conclusion that formic acid destroys proteins, lipids, and polysaccharides in yeast cells, as reported by Zeng et al.^[100]23 . Transcriptome analysis Quality assessment of RNA-seq and genome comparison results Filtered data were obtained after a series of raw data processing steps. The sequence comparison of the filtered high-quality reads with the reference genome showed that the number of clean reads of each sample was above 46.79 M. After data quality control, the mean Q20 values of the control group and the benzoic acid treatment group were 98.44% and 98.40%, respectively, and the mean Q30 values were 95.00% and 94.91%, respectively, which are in line with the requirement that the Q20 and Q30 generally be greater than 85% and 80%, respectively. Assuming that there was no contamination in the experiment and that the reference genome annotation was complete, the total mapped area was generally greater than 65%. For the clean reads produced by Illumina sequencing, the total number of mapped reads was greater than 65% in both the control group and the benzoic acid-treated group. On average, 95.55% and 94.73% coverage of the reference genome was obtained in the control and treatment groups, respectively. The average proportions of uniquely aligned reads were 90.96% and 89.78%, and the average proportions of reads with multiple alignment positions were 4.60% and 4.95%. The sequencing data showed that the quality control data were well compared with the reference genome sequence; that is, the original sequencing data were reliable and could be used for subsequent analysis. Differentially expressed gene screening Differentially expressed genes (DEGs) were screened using |log[2]FC|≥1 and p-adjusted < 0.05 as the screening criteria, and the results are shown in Fig. [101]S1. As shown in Fig. [102]S1, the volcano plot directly shows the overall distribution of DEGs, and the number of significantly downregulated DEGs was greater than that of significantly upregulated DEGs. A total of 1670 DEGs were screened in the benzoic acid treatment group, of which 770 were upregulated and 900 were downregulated. GO enrichment analysis After the DEGs were identified, GO enrichment analysis was performed, as shown in Fig. [103]S2. The GO database divides gene ontologies into three categories: biological process (BP), cell component (CC), and molecular function (MF). As shown in Fig. [104]S2, the DEGs under benzoic acid stress were mainly enriched in the metabolism of purine nucleoside phosphate and purine ribonucleoside monophosphate. This was followed by β-fructofuranase activity, sucrose α-glucosidase activity, cytoplasmic large ribosome subunit, small ribosome subunit, cytoplasmic small ribosome subunit, rRNA ribosome binding, 90 S precursor, ribosome large subunit protein, fructose and mannose transmembrane transport activity, pyruvate decarboxylase activity, oxidoreductase, ribosome, riboprotein complex, ribose body biosynthesis, etc. The results showed that benzoic acid mainly affected nucleotide, ribosome and glucose metabolism in yeast. KEGG pathway enrichment analysis KEGG metabolic pathway enrichment analysis of the screened DEGs was helpful for understanding the stress metabolism pathways activated by cells under benzoic acid stress. Figure [105]9 and Fig. [106]S3 show the KEGG pathway classification statistics and metabolic pathway enrichment maps under benzoic acid stress. Fig. 9. [107]Fig. 9 [108]Open in a new tab KEGG pathway classification of differentially expressed genes under benzoic acid stress. As shown in Fig. [109]9, the metabolic processes of the DEGs under benzoic acid stress that were predominant in the KEGG classification included mainly carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, lipid metabolism, metabolism of other amino acids, energy metabolism, glycan biosynthesis and metabolism, and biosynthesis of other secondary metabolites. Second, in the processing of genetic information, on the main pathways were translation, transcription, folding, classification and degradation, replication and repair. In environmental information processing, membrane transport and signal transduction were the main pathways. In the cell process category, cell growth and death, transport and catabolism were the main pathways involved. In biological systems, cell aging was the main pathway. In Fig. [110]S3, the most frequently annotated DEGs in the KEGG enrichment pathway under benzoic acid stress were associated with ribosomes, followed by starch and sucrose metabolism; purine metabolism; RNA polymerase; galactose metabolism; neomycin, kanamycin and gentamicin biosynthesis; ribosomal biogenesis; arginine and proline metabolism; a carbon pool composed of folic acid; homologous recombination; pyrimidine metabolism; sugar metabolism; thiamine metabolism; valine, leucine and isoleucine biosynthesis; ABC transport; fructose and mannan metabolism; β-alanine metabolism; the Hippo signaling pathway; the biosynthesis of unsaturated fatty acids; and ascorbic acid and aldose metabolism. Real-time fluorescence quantitative PCR validation To verify the reliability of the transcriptomic results, the expression levels of 9 genes (3 upregulated and 6 downregulated genes) in the benzoic acid-treated group and the control group were measured by RT‒qPCR, as shown in Fig. [111]10. The results showed that the expression levels of HXT2 (involved in glycolytic metabolism), PDR15 (involved in plasma membrane ATP-binding transporters), HXK1 (encoding hexokinase), PDC6 (encoding pyruvate decarboxylase subtype), GAL2 (encoding galactose permease) and PDR18 (encoding putative transporters of the ATP-binding box (ABC) family) were downregulated. The expression levels of MNN11 (involved in cell wall synthesis regulation), OPT2 (encoding an oligopeptide transporter) and ERG11 (encoding sterol synthesis) were upregulated. The RT‒qPCR expression data were highly consistent with the transcriptome sequencing data, indicating that the transcriptome sequencing data were reliable. Fig. 10. [112]Fig. 10 [113]Open in a new tab RT‒qPCR results for some differentially expressed genes under benzoic acid stress. Discussion In this paper, benzoic acid was added to analyze its effects on ethanol fermentation and physicochemical characteristics of S. cerevisiae, and the differentially expressed genes in S. cerevisiae cells under 1.2 g/L benzoic acid stress were identified by Illumina RNA Seq technique. It was found that the addition of exogenous benzoic acid could prolong the time taken by yeast cells to enter the rapid growth phase and the stable phase and reduce the final cell biomass. According to the analysis of ethanol fermentation using 250 g/L sucrose, the addition of benzoic acid inhibited the growth and reproduction of cells, reduced cell vitality, slowed the rate of sucrose hydrolysis, and significantly reduced the sugar utilization rate, ethanol production and final fermentation efficiency. To explore the mechanism underlying the toxicity of benzoic acid during ethanol fermentation by S. cerevisiae GJ2008, further analysis of physical and chemical parameters was performed, and the results showed that benzoic acid disrupted cell morphology and that a large number of cavities and folds and intercellular adhesion were visible among the cells. Similar results were also reported in studies on the effects of ferulic acid on S. cerevisiae and Zymomonas mobilis^[114]38. The cell membrane is a selective semipermeable membrane that controls the communication between cells and the external environment^[115]39. Gu et al.^[116]40 explored the mechanism by which phenolic inhibitors damage the cell membrane and cell wall and showed that phenolic acid compounds could easily enter the cell membrane and destroy the integrity of the plasma membrane due to their hydrophobic aromatic ring. Barbieri et al.^[117]41 reported that polyphenols can change the cell membrane potential, and the hydroxyl group of polyphenols can be used as a transmembrane carrier to bring H^+ into the cell and transport K^+ out, while the transport of K^+requires energy consumption, thus affecting the transport of materials and energy between the cell and the external environment. This finding is consistent with the finding that benzoic acid increased the permeability of the cell membrane and caused the cell plasma membrane to rupture, resulting in the leakage of intracellular substances (nucleic acids and proteins) and thus cell death. Yeast cells produce reactive oxygen species in adverse external environments, and excessive reactive oxygen species destroy proteins, lipids, nucleic acids and other biological macromolecules, resulting in DNA damage, enzyme inactivation, lipid peroxidation and other injuries^[118]42–[119]44. The present study showed that yeast cell membrane lipid peroxidation occurs under benzoic acid stress to produce MDA, and the content of MDA increased with increasing stress duration. To further study the response mechanism of yeast cells under benzoic acid stress, changes in the intracellular glycerol content were measured in this study. Yeast cells in the benzoic acid treatment group could cope with the adverse environment by increasing the intracellular glycerol content. FTIR spectroscopy was used to observe changes in the molecular structure and composition of yeast. Benzoic acid treatment changed the levels of proteins, lipids, polysaccharides and other molecular groups, which confirmed that benzoic acid could increase the permeability of the cell membrane, cause the leakage of nucleic acids and proteins, and increase the intracellular MDA content. Under benzoic acid stress, the cell morphology, cell membrane permeability, intracellular substance content, and cell growth and metabolism were significantly affected, which was the direct cause of factors such as high residual sugar content, low ethanol concentration and low fermentation efficiency during ethanol fermentation. To further explore the mechanism underlying the effect of benzoic acid on ethanol fermentation by S. cerevisiae, transcriptomics was used for in-depth analysis. Gene expression related to the ribosome and its bioanabolic pathway The ribosome is the site of protein synthesis in eukaryotes. It is composed of a 40 S subunit, a 60 S subunit and 4 kinds of RNA, which can convert the genetic information contained in the mRNA nucleotide sequence into an amino acid sequence and then synthesize proteins. Ribosomal biogenesis in eukaryotes is an evolutionarily conserved and highly regulated process involving hundreds of transiently bound proteins and RNAs. The synthesis process requires approximately 80 ribosomal proteins and four rRNA species as well as a number of nonribosomal factors, such as snoRNAs and proteins involved in pre-RRNA processing and modification. The synthesis and processing of ribosomal RNA occur to a large extent in a special nuclear structure called the nucleolus. In the nucleolus of S. cerevisiae, rDNA is transcribed to precursor rRNA by RNA polymerase I, which contains mature 18, 5.8, and 25 S rRNA sequences, two external transcription regions (ETSs), and two internal transcription spacer regions (ITSs). 5 S rRNA is surrounded by two nontranscriptional spacers (NTSs) and is transcribed independently by RNA polymerase III^[120]45,[121]46. KEGG enrichment analysis revealed that the expression levels of DEGs involved in ribosomes and the biosynthesis of ribosomes were highly enriched under benzoic acid stress. In eukaryotes, the ribosome as the site of protein synthesis, can recognize the genetic information contained in the mRNA nucleotide sequence, convert the genetic information to amino acid sequence information, and then synthesize proteins, which indicates that benzoic acid affects the process of cell genetic information transmission. There were 145 genes annotated as being related to ribosomes, all of which were upregulated. There were 31 genes annotated to ribosome biosynthesis, 29 of which were upregulated and 2 of which were downregulated. Table [122]2 lists the relevant expression levels of several genes. Table 2. DEGs related to ribosomes and their biosynthesis under benzoic acid stress. Gene ID Gene name Log[2] FC Gene description YBR189W RPS9B 2.39 Ribosome 40 S subunit protein YLR441C RPS1A 1.91 Ribosome 40 S subunit protein YDR450W RPS18A 1.95 Ribosome 40 S subunit protein YJR145C RPS4A 2.08 Ribosome 40 S subunit protein YIL069C RPS24B 2.11 Ribosome 40 S subunit protein YOR293W RPS10A 2.19 Ribosome 40 S subunit protein YOR167C RPS28A 1.94 Ribosome 40 S subunit protein YJL189W RPL39 1.85 Ribosome 40 S subunit protein YER117W RPL23B 2.25 Ribosome 40 S subunit protein YLR325C RPL38 2.29 Ribosome 40 S subunit protein YLL045C RPL8B 2.77 Ribosome 40 S subunit protein YBL092W RPL32 1.73 Ribosome 40 S subunit protein YGL030W RPL30 2.34 Ribosome 40 S subunit protein YNL162W RPL42A 2.12 Ribosome 40 S subunit protein YIL148W RPL40A 2.11 Ubiquitin-ribosome 60 S subunit protein YGL135W RPL1B 1.98 Ribosome 40 S subunit protein YLR409C UTP21 1.11 Production of 18SrRNA and assembly of small ribosome subunits YLR293C GSP1 1.39 GTP binding protein, Maintain nuclear organization, RNA processing and transport YMR093W UTP15 1.72 Nucleolar protein, rRNA processing before 18s YJL069C UTP18 1.07 Small subunit processing protein, rRNA maturation before 18s YDR339C FCF1 1.02 PINc domain nuclease, required for maturation of 18 S rRNA YLR186W EMG1 2.28 rRNA methyltransferase YLR022C SDO1 1.44 Guanine nucleotide exchange Factor (GEF) of Ria1p [123]Open in a new tab Table [124]2 shows that the ribosomal protein (RP) gene, encoding the structural components of ribosomes, and the genes UTP15, UTP18, FCF1 and UTP21, involved in the synthesis and maturation of 18 S rRNA in the nucleolus and the assembly of small subunits of ribosomes, were significantly upregulated, indicating that benzoic acid stress promoted ribosome synthesis and stimulated the biosynthesis of polypeptide chains in yeast cells. The GSP1 gene encodes the Ran GTPase, and the Ran system is composed of small nuclear G proteins and related proteins, which exist in large quantities in eukaryotic cells and are located mainly in the nucleus; these proteins are responsible for transport and communication between the nucleus and cytoplasm^[125]47. In conclusion, the expression of ribosome-related genes was upregulated after the addition of benzoic acid, indicating that yeast cells were stimulated to synthesize more proteins to resist inhibition under benzoic acid stress. Gene expression related to the carbon metabolism pathway Carbon metabolism not only is an important energy donor for maintaining cell vitality but also provides the corresponding precursor substances for other metabolic reactions. Transcriptome data analysis revealed that when benzoic acid inhibitors were added to the medium, the expression of genes involved in starch and sucrose metabolism, glycolysis, fructose and mannose metabolism, ascorbic acid and aldose metabolism, and galactose metabolism significantly decreased, as shown in Table [126]3. Table 3. DEGs associated with carbon metabolism pathways under benzoic acid stress. Metabolic pathways Gene name Log[2] FC Gene description Glycolysis ERR2 −1.13 Enolase ERR3 −1.76 enolase TDH1 −2.41 Glyceraldehyde-3-phosphate dehydrogenase PYK2 −1.09 Pyruvate kinase PDC6 −3.80 Regulate pyruvate kinase activity GLK1 −1.58 glucokinase HXK1 −1.38 Hexokinase isoenzyme I PGM2 −2.56 Glucose phosphate mutase GPM2 −1.02 Convert 3-phosphoglyceric acid to 2-phosphoglyceric acid GPH1 −3.54 Glycogen phosphorylase Starch and sucrose metabolism MAL32 −2.90 Glycogen phosphorylase GSY2 −3.39 Glycogen synthase IMA1 −1.08 Isomaltoolase GSC2 −3.10 Catalytic subunit of 1, 3-glucan synthase TPS1 −1.28 Trehalose-6-P synthase GLC3 −2.21 Glycogen branching enzymes, involved in glycogen accumulation GDB1 −3.45 Glycogen debranching enzyme FKS3 −1.17 Proteins involved in spore wall assembly MAL12 −2.90 Maltase, hydrolyzes disaccharide sucrose SGA1 −1.94 Intracellular sporulation specific glycosylation enzymes Starch and sucrose metabolism GLK1 −1.58 glucokinase TSL1 −1.62 Trehalose 6-phosphate synthase IMA5 −2.50 α-glucosidase IMA4 −2.54 α-glucosidase IMA3 −2.54 α-glucosidase IMA2 −2.92 glucosidase GSY1 −1.43 Glycogen synthase SUC2 −2.32 Sucrose hydrolase ATH1 −1.25 Degradation of intracellular trehalose HXK1 −1.38 Hexokinase isoenzyme I TPS2 −1.11 Trehalose-6-phosphate synthase PGM2 −2.56 Phosphoglucose mutase EXG1 2.71 Cell wall - Glucan assembly Fructose and mannose metabolism GLK1 −1.58 glucokinase SOR1 −2.53 Sorbitol dehydrogenase PFK26 −1.22 phosphofructokinase DSF1 −1.50 Mannose dehydrogenase MAN2 −1.33 Mannose dehydrogenase1 HXK1 −1.38 Hexokinase isoenzymeI FBP26 −1.13 2, 6-diphosphofructokinase Ascorbic acid and alconate metabolism ALD6 −3.54 Cytoplasmic aldehyde dehydrogenase ALD4 −4.65 Mitochondrial aldehyde dehydrogenase ALD5 −1.07 Mitochondrial aldehyde dehydrogenase Hexose transporter family HXT2 −4.74 High-affinity glucose transporters of the superfamily HXT6 −5.42 High affinity glucose transporter HXT7 −5.43 High affinity glucose transporter HXT9 −2.47 Suppose hexose transporter HXT10 −2.87 Suppose hexose transporter HXT11 −1.78 Hexose transporter HXT12 −1.84 Hexose transporter HXT13 −2.07 Hexose transporter HXT15 −1.43 Hexose transporter HXT17 −2.41 Hexose transporter GAL2 −1.23 Galactose permease HXT1 1.30 Low affinity glucose transporter [127]Open in a new tab As shown in Table [128]3, HXK1 (encoding hexokinase, which is the first regulatory enzyme in the EMP pathway and can catalyze glucose, fructose and mannose), PYK2 (encoding pyruvate kinase, which is a rate-limiting enzyme during glycolysis) and PDC6 (regulating the activity of pyruvate kinase) were significantly downregulated under benzoic acid stress, indicating that benzoic acid inhibited the activities of hexokinase and pyruvate kinase, which inhibited the yeast glycolysis process, resulting in insufficient intracellular reducing power and reduced energy synthesis, which was not conducive to alcohol fermentation. GPH1 encodes glycogen phosphorylase, which catalyzes the conversion of glycogen to glucose-1-phosphate and further produce glucose-6-phosphate under the catalytic action of the phosphoglucose mutant enzyme PGM2. The downregulated expression of this gene inhibits the conversion of glucose-1-phosphate to glucose-6-phosphate. SUC2 encodes sucrose hydrolase, a key enzyme in the production of glucose and fructose under sucrose decomposition, and its expression level was significantly downregulated, indicating that benzoic acid inhibited the release of sucrose hydrolase by yeast cells; thus, the sucrose hydrolysis rate in the treatment group was slower than that in the control group. Sugar transporters are key factors in the uptake of sugar by yeast cells. This process is mainly completed by transporters encoded by the HXT gene, which is a member of the major facilitator superfamily (MFS). There are up to 20 sugar transporter genes in S. cerevisiae. Studies have shown that the transcription of sugar transporters in yeast is regulated mainly by the sugar signaling pathway, and some transporters, such as HXT1, are regulated by hyperosmolar glycerol (HOG) metabolism. The proportion of hexose transporters in the plasma membrane of yeast cells is adjusted according to the concentration of available extracellular sugar^[129]48. In this study, the expression levels of glucose transporters encoded by HXT2, HXT6, HXT7, HXT9, HXT10, HXT11, HXT12, HXT13, HXT15, and HXT17 were downregulated. The results showed that benzoic acid reduced the growth rate of yeast cells in the medium with glucose and hexose carbon sources^[130]49. In summary, under benzoic acid stress, the expression of genes encoding sugar transporters was downregulated, the glycolytic pathway was inhibited, and cell energy metabolism was reduced. The HOG metabolic regulation pathway in S. cerevisiae improves the tolerance of yeast cells to benzoic acid. Expression of genes related to the lipid and sterol metabolic pathways The main components of the cell membrane of S. cerevisiaeare glycerophospholipids, sterols and intramembral proteins^[131]50,[132]51. The cell membrane controls the communication between intracellular and external substances and plays an important role in the normal growth and metabolism of cells, as well as the maintenance of a stable intracellular microenvironment and energy metabolism in the cell. Studies have shown that acid stress can cause changes in fatty acid composition, ergosterol content, cell membrane fluidity, cell membrane permeability and cell membrane integrity^[133]52. The fluidity of the cell membrane is closely related to energy conversion, material transport and information transmission. Maintenance of cell membrane fluidity plays an important role in maintaining the physiological function of the cell, thus reducing the damage caused by acid to cells. Studies have shown that under inhibitor stress, the cell membrane structure becomes loose, and the tolerance of yeast cells to inhibitors increases via increased via biosynthesis of long-chain fatty acids^[134]53, which slows down the inhibitory effect of the inhibitor on the cell. As shown in Table [135]4, the expression of the POT1 and POX1 genes involved in the fatty acid β-oxidation process decreased, indicating that energy synthesis was inhibited by benzoic acid. In response to this stress, the expression of the fatty acid synthesis genes SUR4, FEN1 and PHS1, which are related to the cell membrane, increased, indicating that benzoic acid could affect the fluidity and permeability of the membrane by changing the fatty acid content. In addition, the expression levels of the ELO1, SUR4 and FEN1 genes, which encode fatty acid elongases, and the PHS1 gene, which encodes a 3-hydroxyacyl-CoA dehydrase essential for the ER membrane, were significantly upregulated, all of which are involved in the elongation of ultralong-chain fatty acids, suggesting that yeast cells could increase the biosynthesis of long-chain fatty acids to increase their tolerance to benzoic acid stress. Table 4. DEGs associated with lipid metabolism pathways under benzoic acid stress. Metabolic pathways Gene name Log[2] FC Gene description Fatty acid synthesis SUR4 2.13 Prolonging enzymes and facilitate biosynthesis of fatty acids and sphingolipids FEN1 2.12 Fatty acid elongating enzyme, facilitate sphingolipid biosynthesis PHS1 1.35 Facilitate sphingolipid biosynthesis and protein transport, facilitate the elongation of ultra-long chain fatty acids Fatty acid metabolism POT1 −2.65 3-ketoacyl-coA thiolase POX1 −2.66 Fatty acyl-CoA oxidase ELO1 1.83 Catalyzed unsaturated C12-C16 fatty acyl-coA carboxyl terminal extension to C16-C18 fatty acid Sterol synthesis ERG1 1.40 Squalene epoxidase ERG2 1.58 C-8 sterol isomerase ERG3 1.42 C-5 sterol desaturase, a precursor of keratosterol biosynthesis ERG5 1.26 C-22 sterol desaturase, cytochrome P450 enzyme ERG7 1.25 Lanosterol synthase ERG11 1.66 Lanosterol 14-alpha-demethylase, a member of the cytochrome P450 family [136]Open in a new tab The accumulation of ergosterol plays a protective role in yeast under organic acid stress. The ERG1 gene plays a key role in ergosterol-mediated acid tolerance and can prevent staggering and maintain the optimal thickness of the cell membrane to maintain its normal function. Inhibition of the expression of this gene leads to the loss of squalene epoxidase activity^[137]54. In this study, the expression of ERG genes related to ergosterol synthesis was significantly upregulated, indicating that S. cerevisiaeresponds to benzoic acid stress by increasing the ergosterol content. The fact that acid stress induces ergosterol synthesis^[138]55 is consistent with our observations. The ERG gene is related to the ergosterol biosynthesis pathway, which further confirms the important role of ERG1 in ergosterol-mediated acid resistance^[139]56. In addition, multiple genes involved in the ergosterol biosynthesis pathway have been shown to be determinants of the acquisition of tolerance, or they are transcriptionally responsive to acid stress^[140]57. The ERG1 gene encoding squalene epoxide is considered to be the rate-limiting enzyme for ergosterol biosynthesis and catalyzes the epoxidation of squalene to 2,3-oxidized orangene. Taken together, these results provide important insight into the key genes involved in ergosterol synthesis and their role in improving the tolerance of S. cerevisiae to benzoic acid. Gene expression related to nucleotide metabolism There are two pathways for the synthesis of purine nucleotides in S. cerevisiae: de novo synthesis and remediation. Studies have shown that excessive adenine enters the cell to form AMP through the remedial pathway, and the AMP is further converted to ADP and ATP. However, when ADP and ATP are present in excess, they inhibit the activity of the aminophosphoribose transferase encoded by ADE4 in the de novo synthesis pathway^[141]58. The homeostasis of AMP, ADP, and ATP plays an important role in maintaining cellular energy and nucleotide metabolism, and interconversion among these substances is controlled by the highly conserved ADK1-encoded adenylate kinase^[142]59. The PRS1, PRS2, PRS3, PRS4 and PRS5 genes encode PRPP synthetases, and PRPP is involved in several metabolic pathways involving purine nucleotides. Studies have shown that loss of PRS1 and PRS3 greatly affect cell metabolism, while loss of PRS4 has little effect on cells^[143]60,[144]61. Ji et al. reduced cyclic adenosine phosphate production by 3.85% through the overexpression of ADK1. Overexpression of the PRS1, PRS3 and ADE4 genes increased cyclic adenosine monophosphate production by 9.03%, 4.17% and 6.06%, respectively. As shown in Table [145]5, genes involved in the regulation of purine metabolism, namely, ADE1, ADE2, ADE4, ADE6, ADE8, ADE12, ADE13 and ADE17, were significantly upregulated, and the overexpression of ADE1, ADE13 and ADE17 increased the intracellular ATP content and the activity of the antioxidant enzyme SOD^[146]62. This suggests that the purine nucleic acid biosynthesis pathway activity may increase in S. cerevisiae in response to an insufficient intracellular energy supply under benzoic acid stress. Table 5. DEGs related to nucleotide metabolism pathways under benzoic acid stress. Gene ID Gene name Log[2] FC Gene description YOR128C ADE2 2.35 Phosphoribose aminoimidazole carboxylase catalyzes “de novo” purine nucleotide biosynthesis YMR120C ADE17 2.67 Purine biosynthesis “de novo” enzymes YNL220W ADE12 1.04 Catalyze the synthesis of adenosine monophosphate from inosine 5’ monophosphate during purine nucleotide biosynthesis YMR300C ADE4 3.29 Aminophosphoribose transferase YLR359W ADE13 2.08 Catalyze “de novo” purine nucleotide biosynthesis YDR408C ADE8 1.72 Phosphoribose cinnamamide transformylase YGR061C ADE6 1.84 formylglycine-nucleotide-synthase YAR015W ADE1 2.83 N-succinyl-5-aminoimidazole-4-carboxyamide nucleotide synthetase YDR399W HPT1 1.48 Catalyze the transfer of the phosphoribosyl portion of 5-phosphoribosyl-α−1 pyrophosphate to the purine base to form pyrophosphate and purine nucleotides YDL166C FAP7 1.11 An essential NTPase for the synthesis of small ribosome subunits YOL061W PRS5 1.43 PRPP for synthesis of nucleotides, histidine and tryptophan biosynthesis YOR347C PYK2 −1.09 Pyruvate kinase YHL011C PRS3 1.26 5-phospho-ribose 1(α) -pyrophosphate synthetase YDR226W ADK1 1.16 Adenylate kinase YKL181W PRS1 1.36 5-phospho-ribose 1(α) -pyrophosphate synthetase YJR010W MET3 −1.29 ATP sulfase, involved in methionine metabolism [147]Open in a new tab Conclusion To explore the mechanism underlying the toxic effect of benzoic acid in sugarcane molasses on Saccharomyces cerevisiae cells, in this study, the effect of exogenous addition of benzoic acid on cells was examined. The results showed that benzoic acid affected the growth and fermentation metabolism of S. cerevisiae, resulting in a high residual sugar content, low ethanol concentration and low fermentation efficiency. Further analysis of physical and chemical properties revealed that benzoic acid disrupted cell morphology, damaged the integrity of the cell wall and cell membrane, increased the permeability of the cell membrane and extravasated intracellular matter (nucleic acid and protein), increased the MDA content, and caused severe cell injury and even death. In response to external stress, yeast cells balance the intracellular osmotic pressure by increasing the intracellular glycerol content. Transcriptomic sequencing was used to analyze the response mechanism of S. cerevisiae under benzoic acid stress. The results showed that the expression of genes associated with ribosomes and their biosynthesis, RNA polymerase, lipid metabolism, nucleic acid metabolism and cofactor synthesis was significantly upregulated after benzoic acid treatment. The expression of genes related to the glycolytic pathway, carbon metabolism and thiamine metabolism was significantly downregulated. These results indicated that benzoic acid inhibited the glycolysis process in yeast and reduced the absorption and utilization of sugar and the ATP energy supply. In response to benzoic acid stress, S. cerevisiae upregulated genes related to ribosomal biosynthetic metabolic pathways and promoted the synthesis of macromolecular substances and proteins. On the other hand, the expression of ELO1, SUR4, FEN1 and ERG1 was upregulated, which led to the extension of long-chain fatty acids and the accumulation of ergosterol to maintain cell membrane structure. In summary, this paper studied the effects of benzoic acid on the fermentation process and physicochemical properties of S. cerevisiae, and analyzed the response mechanism of S. cerevisiae under benzoic acid stress by transcriptome sequencing technology, so as to comprehensively analyze the toxic effects of benzoic acid on yeast cells. According to the transcriptomic data, the stress resistance genes under benzoic acid stress can be targeted, which provides a theoretical basis for the future use of CRISPR molecular biology technology to perform gene editing such as knocking out or modifying genes related to benzoic acid sensitivity or introducing specific genes from other strains with high tolerance, obtain tolerance strains through overexpression, improve ethanol fermentation ability, and realize the production of high concentration ethanol by molasses. Electronic supplementary material Below is the link to the electronic supplementary material. [148]Supplementary Material 1^ (513.8KB, docx) Author contributions Xiu-Feng Long, Yu-lei Xu and Xue-mei Zhao designed the experiments. Yu-lei Xu and Xue-mei Zhao performed the experiments. Xiu-Feng Long, Yu-lei Xu and Xue-mei Zhao analyzed the data and wrote the paper. All of the authors reviewed the paper. Funding This work was funded by the National Natural Science Foundation of Guangxi Province (No. 2024GXNSFBA010351), the Opening Project of Guangxi Key Laboratory of Green Processing of Sugar Resources (No. GXTZYZR202206) and Guangxi University of Science and Technology Doctoral Fund (No. 20Z18). Data availability The sequence data in this study has been deposited in the NCBI database under the BioProject accession number PRJNA1144383. All the other data that support the findings of this study are available from the corresponding author upon reasonable request. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References