Growth inhibition by acetate is a major stress that is normally encountered during fermentation with lignocellulose feedstocks. Acetate is released from acetylated xylose residues in hemicellulose during pre-treatment, and can exceed 10 g/L in corn stover hydrolysate. Consequently, developing a recombinant Z. mobilis strain that has higher resistance to acetate is on the critical path for commercialization of cellulosic ethanol. To identify genes that confer acetate tolerance, a ZW801-4 genomic knockout library (consisting of >18, 000 independent mutants) was screened for better growth on acetate; ZW801-4 is a xylose-utilizing Z. mobilis strain. After seven rounds of the mutant enrichment process, single colonies were obtained for DNA sequence analysis. Remarkably, all of the colonies examined had a transposon insert in a single ORF, one that shows homology to a global regulator of gene expression in E. coli and other gram negative bacteria. Furthermore, three different insertion events were identified. During this talk we will demonstrate that intentional inactivation of this gene does increase acetate-resistance. This simple genetic manipulation also improves fermentation performance in concentrated mixtures of glucose and xylose when acetate is present, especially during the late stage of fermentation when the stress level is the highest.