Abstract Background High temperature and ethanol are two critical stress factors that significantly challenge bioethanol production using Saccharomyces cerevisiae. In this study, the tolerance mechanisms of the multi-tolerant S. cerevisiae strain E-158 to heat stress and combined heat-ethanol stress were investigated using comparative transcriptomics. Results Under heat stress at 44 °C, glucose transport and reactive oxygen species (ROS) scavenging were significantly upregulated, while gluconeogenesis, acetate formation, and dNDP formation showed significant downregulation. Under combined heat (43 °C) and ethanol (3% v/v) stress, glucose transport, glycolysis, acetate formation, peroxisome activity, ROS scavenging, and ribosome synthesis were significantly upregulated, while glycerol formation, cellular respiration and dNDP formation exhibited significant downregulation. Fourteen transcription factors (TFs), considered to play a key role in both stress conditions, were individually overexpressed and deleted in S. cerevisiae strain KF-7 in this study. Among these TFs, Gis1p, Crz1p, Tos8p, Yap1p, Dal80p, Uga3p, Mig1p, and Opi1p were found to contribute to enhanced heat tolerance in S. cerevisiae. Compared with KF-7, strains overexpressing DAL80 and CRZ1 demonstrated markedly improved fermentation performance under stress conditions. Under heat stress at 44 °C, glucose consumption increased by 10% and 12%, respectively, for strains KF7DAL80 and KF7CRZ1, while ethanol production increased by 12% and 15%, respectively, compared to KF-7. Under combined stress conditions of 43 °C and 3% (v/v) ethanol, glucose consumption increased by 67% and 44%, ethanol production by 116% and 77%, and ethanol yield by 29% and 22%, respectively, for KF7DAL80 and KF7CRZ1 compared to KF-7. KF7CRZ1 performs comparably to E-158, while KF7DAL80 outperforms E-158. Conclusions This study provides valuable theoretical insights and identifies critical TF targets, contributing to the development of robust S. cerevisiae strains for improved bioethanol production. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-025-02653-2. Keywords: Saccharomyces cerevisiae, Comparative transcriptome, Heat tolerance, Combined heat and ethanol stress, Transcription factors Background Bioethanol, characterized by its transportability, high energy density, and low greenhouse gas emissions, is considered a highly promising liquid fuel for low-carbon transportation [[32]1]. Saccharomyces cerevisiae, known for its excellent ethanol production capacity and good tolerance to various stresses, has traditionally been the preferred strain for fuel ethanol production [[33]2]. However, in industrial ethanol production, yeast cells face multiple stresses, such as high temperatures and elevated ethanol concentrations [[34]3, [35]4]. These stress conditions can inhibit yeast growth, causing delays or complete stalls in fermentation, thereby significantly hindering industrial productivity. Moreover, to save time and reduce costs in the production of ethanol from lignocellulosic and starch-based feedstocks, the industry commonly employs simultaneous saccharification and fermentation (SSF) [[36]5, [37]6]. There is a significant difference between the optimal temperature for enzymatic hydrolysis (45–50 °C) and that for fermentation (30–35 °C), leading to increased enzyme usage and higher cooling costs. Enhancing the growth and fermentation performance of S. cerevisiae under high-temperature conditions would, therefore, greatly benefit the SSF process. While S. cerevisiae has evolved certain mechanisms to tolerate individual stress, enhancing its tolerance to multiple concurrent stresses remains a critical challenge in industrial applications [[38]7, [39]8]. Addressing this issue could significantly enhance the efficiency and cost-effectiveness of bioethanol production processes. Several studies have attempted to isolate strains with thermotolerance from natural environments. Pandey et al. [[40]9] isolated a S. cerevisiae strain, NGY10, from sugarcane distillery waste, which produced 46.81 g/L of ethanol during fermentation at 40 °C with an initial glucose concentration of 100 g/L. Auesukaree et al. [[41]10] isolated a thermotolerant S. cerevisiae strain from tropical fruits that produced 38 g/L of ethanol under fermentation conditions of 41 °C and an initial glucose concentration of 100 g/L. Despite these achievements, pursuing higher thermal tolerance remains crucial for ensuring enzymatic activity and fermentation efficiency. Therefore, further improving the high-temperature tolerance of cells is indispensable. To identify potential targets for improving tolerance, many researchers have employed omics technologies to elucidate the stress–response mechanisms of S. cerevisiae strains to high temperatures, ethanol, and other stresses. Yang et al. [[42]11] obtained an ethanol-tolerant mutant, YN81, using ultraviolet–diethyl sulfate (UV–DES) mutagenesis. Through comparative transcriptomics, they highlighted the importance of membrane-associated genes for ethanol tolerance. Gan et al. [[43]12] knocked out 23 transcription factors (TFs) and identified three key TFs associated with thermotolerance: Sin3p, Srb2p, and Mig1p. However, current research still has limitations. Many studies have focused on single-stress conditions, whereas fermentation processes typically involve multiple concurrent stresses. Furthermore, the strains used in these studies often have weak inherent tolerance to stresses, including ethanol, heat, and toxic inhibitors, which makes their response mechanisms and gene targets less relevant for practical applications. Understanding the response mechanisms of multi-stress-tolerant S. cerevisiae strains under multiple stress conditions would provide more valuable guidance for constructing robust strains suitable for industrial production. In our earlier work, a multi-tolerant industrial S. cerevisiae strain, E-158, was developed [[44]13]. E-158 exhibited superior tolerance to five stress conditions: high temperature, high ethanol concentration, combined heat and ethanol stress, high sugar concentration, and high salt concentration [[45]13]. A comparative transcriptome analysis of strain E-158 and its original strain, KF-7, under five stress conditions identified 28 shared differentially expressed genes (DEGs) [[46]14]. The overexpression of CRZ1 and ENA5, along with the deletions of ASP3, YOL162W, YOR012W, and TOS8 in strain KF-7, was found to significantly enhance tolerance to multiple stress conditions [[47]14]. In the present study, a detailed comparative transcriptome analysis was performed under two stress conditions: high temperature (44 °C) and combined heat-ethanol stress (43 °C and 3% v/v ethanol). The study aims to identify key TFs involved in thermotolerance and dual-stress tolerance to heat and ethanol. These findings provide valuable theoretical insights and offer promising targets for the development of robust strains specifically tailored for industrial bioethanol production. Methods Strains and media All strains used in this study are listed in Table [48]1. The flocculating diploid industrial S. cerevisiae strain KF-7 was used as the original strain [[49]15]. The multi-tolerant strain E-158 was derived from KF-7 [[50]13]. E. coli DH5α (Takara Bio Inc., Japan) was used for gene cloning and manipulation. Table 1. Saccharomyces cerevisiae strains used in this study Strains Description References