Microbial production of fuels and chemicals from lignocellulosic biomass provides promising bio-renewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocellulosic biomass materials are glucose and xylose. While industrial Escherichia coli strains efficiently utilize glucose, their ability to utilize xylose is often repressed in the presence of glucose. Here we independently evolved three E. coli strains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into the E. coli D-lactate producer TG114, 94 % of a glucose-xylose mixture (50 g L-1 each) was utilized in mineral salt media that led to a 50 % increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in different E. coli hosts, including wild-type, suggesting its potential wide application in industrial E. coli biocatalysts.