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Background Strong multiple stress-tolerance is a desirable characteristic for  Saccharomyces cerevisiae  when different feedstocks are used for economical industrial ethanol production. Random mutagenesis or genome shuffling has been applied for improving multiple stress-tolerance, however, these techniques are generally time-consuming and labor cost-intensive and their molecular mechanisms are unclear. Genetic engineering, as an efficient technology, is poorly applied to construct multiple stress-tolerant industrial  S. cerevisiae  due to lack of clear genetic targets. Therefore, constructing multiple stress-tolerant industrial  S. cerevisiae  is challenging. In this study, some target genes were mined by comparative transcriptomics analysis and applied for the construction of multiple stress-tolerant industrial  S. cerevisiae  strains with prominent bioethanol production. Results Twenty-eight shared differentially expressed genes (DEGs) were identified by comparative analysis of the transcriptomes of a multiple stress-tolerant strain E-158 and its original strain KF-7 under five stress conditions (high ethanol, high temperature, high glucose, high salt, etc.). Six of the shared DEGs which may have strong relationship with multiple stresses, including functional genes ( ASP3 ,  ENA5 ), genes of unknown function ( YOL162W ,  YOR012W ), and transcription factors (Crz1p, Tos8p), were selected by a comprehensive strategy from multiple aspects. Through genetic editing based on the CRISPR/Case9 technology, it was demonstrated that expression regulation of each of these six DEGs improved the multiple stress-tolerance and ethanol production of strain KF-7. In particular, the overexpression of  ENA5  significantly enhanced the multiple stress-tolerance of not only KF-7 but also E-158. The resulting engineered strain, E-158-ENA5, achieved higher accumulation of ethanol. The ethanol concentrations were 101.67% and 27.31% higher than those of the E-158 when YPD media and industrial feedstocks (straw, molasses, cassava) were fermented, respectively, under stress conditions. Conclusion Six genes that could be used as the gene targets to improve multiple stress-tolerance and ethanol production capacities of  S. cerevisiae  were identified for the first time. Compared to the other five DEGs,  ENA5  has a more vital function in regulating the multiple stress-tolerance of  S. cerevisiae . These findings provide novel insights into the efficient construction of multiple stress-tolerant industrial  S. cerevisiae  suitable for the fermentation of different raw materials. Graphical Abstract

biotechnology for biofuels and bioproducts

Screening novel genes by a comprehensive strategy to construct multiple stress-tolerant industrial Saccharomyces cerevisiae with prominent bioethanol production