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Background  The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable  Saccharomyces   cerevisiae  strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial  S.   cerevisiae  strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance ( ARI1 ,  PAD1  or  TAL1 ) coupled with an inhibitor-specific gene ( ADH6 ,  FDH1  or  ICT1 ). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor.    Results  The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g.  TAL1  +  FDH1 ). The  TAL1  +  ICT1  combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the  ICT1  protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes.    Conclusions  The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13068-021-02021-w.

Rational engineering of Saccharomyces cerevisiae towards improved tolerance to multiple inhibitors in lignocellulose fermentations