Background

Succinic acid (SA), a C-4 building block chemical, has been widely used in medicine, agriculture, industry, and so on [1]. For example, succinate is widely used as an intermediary feedstock to produce chemicals such as 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, succinate salts, and adipic acid [2]. In 2004, the U.S. Department of Energy (DOE) proposed that SA is one of the five most promising bio-based platform chemicals.

The production of SA via petrochemical processing is facing challenges posed by unsustainable fossil energy supplies and increased environmental burdens. Therefore, microbial factories have become a promising alternative. Microorganisms such as Actinobacillus succinogenes [32, 34, 63, 64]. An increased PQC activity might enhance the SA tolerance of S. cerevisiae. However, NBRC2018 showed a significant downregulation of HSP26, which could potentially undermine its ability to tolerate SA stress. Given that HSP26 and HSP42 encode the only two sHsps in S. cerevisiae, they might share similar functions in responding to SA stress. Therefore, to verify their roles in SA tolerance, we overexpressed HSP26 and HSP42 in all three strains. The constitutively strong promoter TEF1p was used to express HSP26 and HSP42. Six strains were obtained and named as KF7 + TSP26, KF7 + TSP42, N19 + TSP26, N19 + TSP42, N20 + TSP26, and N20 + TSP42, respectively. The growths of these strains under different concentrations of SA (0 g/L, 60 g/L, and 80 g/L) were compared with their original strains (Fig. 6; Table 4).

Fig. 6
figure 6

Influence of HSP26 or HSP42 overexpression on the SA tolerances of S. cerevisiae strains KF7 (a), NBRC1958 (b), and NBRC2018 (c). Cells were exposed to 0 g/L, 60 g/L, and 80 g/L SA, respectively. The initial inoculum was OD600 0.4. OD600 was measured at 10 h of cultivation. Taking the original strains KF7, NBRC1958, and NBRC2018 as controls for each group. Values and standard deviations were calculated from three repeated samples. *p < 0.05; **p < 0.001; ***p < 0.0001; ****p < 0.00001; ns, no statistically significant difference

Full size image
Table 4 The growth enhancement ratios (%) of the engineered strains compared to its original strain under different concentrations of SA
Full size table

The overexpression of HSP26 and HSP42 led to a significant improvement in the growth of NBRC1958. In detail, when HSP26 was overexpressed, the cell growth of NBRC1958 increased by 50%, 19%, and 52% at 0 g/L, 60 g/L, and 80 g/L SA, respectively. Overexpression of HSP42 had a nearly identical effect on the cell growth of NBRC1958. The results indicated that enhancing the activity of sHsps can elevate the intrinsic growth capacity of NBRC1958, thus boosting its ability to grow under SA stress. This finding aligns with prior research, which demonstrated that sHsps serve as universal effectors of longevity, and the overexpression of HSP26 extended the replicative lifespan of yeast cells [65]. Consistently, sHsps have been implicated in S. cerevisiae’s response to other weak acids, such as sorbic acid and citric acid [66, 67]. Overexpression of HSP26 has been shown to enhance the strains’ tolerance to sorbic acid [66]. However, for the other two strains, neither the overexpression of HSP26 nor HSP42 had a notable effect on cell growth. It was likely that some other key limiting factors may play a more decisive role in determining the SA tolerance of the two strains. In conclusion, overexpressing HSP26 or HSP42 in strains with inherent low sHsps activities is one of the methods to improve SA tolerance.

Genetic background has been proven to affect sugar metabolism and inhibitor tolerance of S. cerevisiae [68, 69]. In this study, we observed that the response mechanisms of different strains to SA were indeed influenced by their genetic backgrounds (Figs. 3, 4 and 5). For example, KF7 and NBRC2018 showed notable differences in the regulation of acetyl-CoA metabolism under SA stress. When KF7 was exposed to SA, genes associated with fatty acids beta-oxidation and the glyoxylate cycle were significantly downregulated. As a result, the production of acetyl-CoA from peroxisomes reduced, leading to a correspondingly reduction in endogenous succinate synthesis. Instead, exogenous SA was likely to be used for acetyl-CoA biosynthesis to maintain intracellular acetyl-CoA levels in KF7 (Fig. 3). On the contrary, when NBRC2018 encountered severe SA stress (60 g/L), genes related to fatty acid β-oxidation and the glyoxylate cycle were significantly upregulated. Despite the increased availability of acetyl-CoA, this upregulation also promoted the generation of endogenous succinate, which further exacerbating the intracellular SA stress. This differential response may be one of the main reasons why NBRC2018 was less tolerant to SA than KF7. Thus, when develo** SA-producing strains, it is crucial to carefully consider the intracellular acetyl-CoA levels and the strains’ capacity to utilize SA.

In general, weak acids typically cause toxicity in S. cerevisiae cells through several mechanisms, include intracellular acidification, membrane damage, oxidative stress, protein aggregation, carbon metabolism disruption, etc. [70]. Accordingly, yeast cells employed a variety of complex and diverse regulatory mechanisms to cope with weak acids [70, 71]. For instance, the activities of plasma membrane H+-ATPase and ABC transporters are increased in response to acetic acid [71]; the maintenance of CWI is crucial for yeast’s adaptation and tolerance to acetic and lactic acid [72]; the biosynthesis of purine and methionine is reduced in response to formic and acetic acids [73,

Conclusion

In this study, five S. cerevisiae strains with different genetic backgrounds were compared for their SA tolerances. KF7 and NBRC1958 with excellent SA tolerances, and NBRC2018 with poor SA tolerance, were selected to investigate the response mechanisms of S. cerevisiae to SA through comparative transcriptomic analysis. Few genes were significantly regulated under 20 g/L SA in three strains. When exposed to 60 g/L SA, the three strains showed different response mechanisms. Overall, the DEGs were involved in carbon metabolism, amino acid metabolism, protein folding, meiosis, membrane proteins and cell wall structure. We conclude that the genetic background of the host strain is important for the construction of good SA producing strains. The inherent activities of sHsps, acetyl-CoA levels and the potential SA consumption capacity of the host strains must be considered. This study provides theoretical guidance and tolerant strains for the breeding of robust SA-producing strains.