Display omitted •Flocculation leads to profound changes of protein kinases under acetic acid stress.•Phosphoproteome analysis identified key kinases responded to acetic acid stress.•Overexpression of HOG1 and GPX1 reduced ROS content and improved stress tolerance.•Overexpression of protein kinase Akl1 enhanced ethanol fermentation using corn cob. Cell self-flocculation endows yeast strains with improved environmental stress tolerance that benefits bioproduction. Exploration of the metabolic and regulatory network differences between the flocculating and non-flocculating cells is conducive to developing strains with satisfactory fermentation efficiency. In this work, integrated analyses of transcriptome, proteome, and phosphoproteome were performed using flocculating yeast Saccharomyces cerevisiae SPSC01 and its non-flocculating mutant grown under acetic acid stress, and the results revealed prominent changes in protein kinases. Overexpressing the mitogen-activated protein kinase Hog1 upregulated by flocculation led to reduced ROS accumulation and increased glutathione peroxidase activity, leading to improved ethanol production under stress. Among the seven genes encoding protein kinases that were tested, AKL1 showed the best performance when overexpressed, achieving higher ethanol productivity in both corncob hydrolysate and simulated corn stover hydrolysate. These results provide alternative strategies for improving cellulosic ethanol production by engineering key protein kinases in S. cerevisiae.