In Klebsiella pneumoniae, aldehyde dehydrogenases (ALDH) convert 3-hydroxypropionaldehyde (3-HPA) into 3-hydroxypropionic acid (3-HP). Although ALDHs can increase the production of 3-HP in K. pneumoniae, the substrate specificity of ALDH homologues from other microorganisms toward 3-HPA is less documented. Here we report that DhaS, a putative ALDH from Bacillus subtilis, shows high specificity toward 3-HPA when heterologously expressed in K. pneumoniae. Using NAD(+) as a cofactor, DhaS exhibited higher catalytic activity (2.3 U mg(-1)) and lower K (m) value (0.4 mmol l(-1)) toward 3-HPA than that toward other aldehydes. Under shake-flask conditions, the recombinant strain produced 2.1 g 3-HP l(-1) in 24 h, which is 3.9-fold of that in a control harboring a blank vector. Under non-optimized bioreactor conditions, the recombinant strain produced 18 g 3-HP l(-1) and 1,3-propanediol (1,3-PDO) at 27 g l(-1) in 24 h. The overall conversion rate from glycerol to 3-HP and 1,3-PDO reached 59.4 mol mol(-1). Homology modeling of DhaS illustrates substrate specificity and NAD(+)-binding site. DhaS is thus a 3-HPA-specific enzyme useful for production of 3-HP.