During the industrial production of ethanol using yeast, the cells are exposed to stresses that affect their growth and productivity; therefore, stress-tolerant yeast strains are highly desirable. To increase ethanol production from glycerol, a greater tolerance to osmotic and ethanol stress was engineered in yeast strains that were impaired in endogenous glycerol production by the overexpression of both SPT3 and SPT15, components of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex. The engineered strain YPH499fps1 Delta gpd2 Delta (pGcyaDak, pGupSpr3.15Cas) formed significantly more biomass compared to the strain YPH499fps1 Delta gpd2 Delta (pGcyaDak, pGupCas), and both engineered strains displayed increased biomass when compared to the control YPH499fps1 Delta gpd2 Delta (pESC-TRP) strain. The trehalose accumulation and ergosterol content of these strains were 2.3-fold and 1.6-fold higher, respectively, than the parent strains, suggesting that levels of cellular membrane components were correlated with the enhanced stress tolerance of the engineered strains. Consequently, the. ethanol production of the engineered strain YPH499fps1 Delta gpd2 Delta (pGcyaDak, pGupSpt3.15Cas) was 1.8-fold more than that of strain YPH499fps1 Delta gpd2 Delta (pGcyaDak, pGupCas), with about 8.1 g/L ethanol produced. In conclusion, we successfully established that the co-expression of SPT3 and SPT15 that improved the fermentation performance of the engineered yeast strains which produced higher ethanol yields than stress-sensitive yeast strains. (c) 2012 Elsevier Inc. All rights reserved.