3-Hydroxypropionic acid (3-HP) is a value-added chemical for polymer synthesis. For biosynthesis of 3-HP from glycerol, two dhaB and dhaR clusters encoding glycerol dehydratase and its reactivating factor, respectively, were cloned from Lactobacillus brevis KCTC33069 and expressed in Escherichia coli. Coexpression of dhaB and dhaR allowed the recombinant E. coil to convert glycerol to 3-hydroxypropion-aldehyde, an intermediate of 3-HP biosynthesis. To produce 3-HP from glycerol, fed-batch fermentation with a two-step feeding strategy was designed to separate the cell growth from the 3-HP production stages. Finally, E. coli JHS00947 expressing L.brevis dhaB and dhaR, and E. coil aldH produced 14.3 g/L 3-HP with 0.26 g/L-h productivity, which were 14.6 and 8.53 times higher than those of the batch culture. In conclusion, overexpression of L. brevis dhaB and dhaR clusters and E. coil aldH, and implementation of the two-step feeding strategy enabled recombinant E. coli to convert glycerol to 3-HP efficiently. (C) 2012 Elsevier Ltd. All rights reserved.