Escherichia coli expressing the Lactobacillus brevis dhaB(1)B(2)B(3) and dhaR(1)R(2) clusters and Pseudomonas aeruginosa aldhH was engineered to produce 3-HP from glucose and xylose via the glycerol biosynthetic pathway. Glycerol, a key precursor for 3-HP biosynthesis was produced by overexpression of the GPD1 and GPP2 genes from Saccharomyces cerevisiae. For relief of carbon catabolite repression, deletion of the chromosomal ptsG gene and overexpression of the endogenous xylR gene rendered engineered E. coli JHS01300/pCPaGGRm to utilize glucose and xylose simultaneously and to produce glycerol at 0.48 g/g yield and 0.35 g/L-h productivity. Finally, engineered E. coli JHS01300/pELDRR + pCPaGGRm produced 29.4 g/L of 3-HP with 0.54 g/L-h productivity and 0.36 g/g yield in a sugar-limited fed-batch fermentation. It was concluded that dual modulation of sugar transport and glycerol biosynthesis is a promising strategy for efficient conversion of glucose and xylose to 3-HP. (C) 2015 Elsevier Ltd. All rights reserved.