Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coil in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coil and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coil MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coil was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing. (C) 2011 Elsevier Inc. All rights reserved.