Sugar derived from biomass is usually a mixture of glucose and other sugars. When mixed sugars are fed to Escherichia coli, glucose is preferentially utilized while other sugars remain unutilized. This phenomenon is known as carbon catabolite repression (CCR). To utilize mixed sugars effectively, we isolated a new E. coli mutant that is negative for CCR. The mutant strain was revealed to have a nucleotide substitution at the promoter region of mlc encoding a global transcriptional repressor for carbohydrate metabolism. The identified mutation, named mlc∗, was a promoter-up type, and the mlc∗ promoter exhibited 17-fold higher activity than the wild-type mlc promoter. Therefore, the mlc∗ mutation causes Mlc overexpression and a shortage of PtsG, which is a glucose-specific permease that is repressed by Mlc. The disruption of ptsG (ΔptsG) is known to induce a CCR-negative phenotype; the mlc∗ strain also exhibits the same phenotype via the same mechanism. As a sample application of the mlc∗ strain, we produced isobutanol from mixed sugars. Using glucose-xylose mixed sugar, the mlc∗ strain produced 1.4-fold more isobutanol than the parental wild-type strain. Also, the mlc∗ strain produced similar or greater amounts of isobutanol than other CCR-negative strains, ΔptsG and crp∗ (crp∗, encoding the constitutive-active mutant of cAMP receptor protein). In conclusion, the mlc∗ strain is a new CCR-negative strain that is useful for producing valuable compounds from mixed sugars.