AIMS: The impact of promoter selection on the overexpression of the XKS1 gene in Saccharomyces cerevisiae is investigated with a focus on optimizing xylose metabolism for second-generation ethanol production. The goal was to identify how different promoters affect the fermentation performance of laboratory and industrial yeast strains under various media conditions. METHODS AND RESULTS: Four constitutive promoters-TEF1p, ADH1p, PGK1p, and TDH3p-were tested to overexpress XKS1 in two strains of S. cerevisiae, one laboratory strain (BY4742) and one industrial strain (PE-2B), both engineered with a heterologous xylose isomerase pathway. The strains were evaluated in defined (YNB) and complex (YPDX) media, as well as a synthetic sugarcane hydrolysate, over a 144-hour fermentation period. Promoter choice significantly influenced cell growth, xylose consumption and ethanol production. In the laboratory strain, TEF1p yielded the highest ethanol production in YPDX, while TDH3p promoted higher biomass formation. In the industrial strain, ADH1p, TEF1p, and PGK1p led to high ethanol yields in YPDX, with ADH1p showing superior performance in the synthetic hydrolysate. RT-qPCR reveals lower XKS1 expression levels render a better trait for BY4742, while the opposite is observed for PE-2B. CONCLUSIONS: It is demonstrated that promoter selection is crucial for optimizing XKS1 expression and xylose metabolism in S. cerevisiae. Promoters must be carefully tailored to the yeast strain and fermentation conditions to maximize ethanol production, providing strategic insights for enhancing the industrial fermentation of lignocellulosic biomass.