Escherichia coli was metabolically engineered for the production of D-ribose, a functional five-carbon sugar, from xylose. For the accumulation of D-ribose, two genes of transketolase catalyzing the conversion of D-ribose-5-phosphate to sedoheptulose-7-phosphate in pentose phosphate pathway were disrupted to create a transketolase-deficient E. coli SGK013. In batch fermentation, E. coli SGKO13 grew by utilizing glucose and then started to produce D-ribose from xylose after glucose depletion. E. coli SGK013 produced 0.75 g/L of D-ribose, which was identical to the standard D-ribose as confirmed by HPLC and LC/MS analyses. To improve D-ribose production, the ptsG gene encoding the glucose-specific IICB component was disrupted additionally, resulting in the construction of E. coli SGK015. The carbon catabolite repression-negative E. colt SGK015 utilized xylose and glucose simultaneously and produced up to 3.75 g/L of D-ribose, which is a 5-fold improvement compared to that of E. colt SGK013. (C) 2016 Elsevier Ltd. All rights reserved.