As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineeredEscherichia coliusing glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, thedhaB1234andgdrABgenes fromKlebsiella pneumoniaeencoding glycerol dehydratase and glycerol reactivase, respectively, were introduced intoE. colito convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, theydcWgene fromK. pneumoniaeencoding gamma-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of theydcWgene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineeredE. colistrain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g center dot L-1 center dot h(-1), respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.