2-Deoxy-d-ribose 5-phosphate aldolase (DERA) accepts a wide variety of aldehydes and is used in de novo synthesis of 2-deoxysugars, which have important applications in drug manufacturing. However, DERA has low preference for non-phosphorylated substrates. In this study, DERA from Klebsiella pneumoniae (KDERA) was mutated to increase its enzyme activity and substrate tolerance towards non-phosphorylated polyhydroxy aldehyde. Mutant KDERA(K12) (S238D/F200I/Delta Y259) showed a 3.15-fold improvement in enzyme activity and a 1.54-fold increase in substrate tolerance towards d-glyceraldehyde compared with the wild type. Furthermore, a whole-cell transformation strategy using resting cells of the BL21(pKDERA12) strain, containing the expressed plasmid pKDERA12, resulted in increase in 2-deoxy-d-ribose yield from 0.41 mol/mol d-glyceraldehyde to 0.81 mol/mol d-glyceraldehyde and higher substrate tolerance from 0.5 to 3 M compared to in vitro assays. With further optimization of the transformation process, the BL21(pKDERA12) strain produced 2.14 M (287.06 g/L) 2-deoxy-d-robose (DR), with a yield of 0.71 mol/mol d-glyceraldehyde and average productivity of 0.13 mol/L center dot h (17.94 g/L center dot h). These results demonstrate the potential for large-scale production of 2-deoxy-d-ribose using the BL21(pKDERA12) strain. Furthermore, the BL21(pKDERA12) strain also exhibited the ability to efficiently produce 2-deoxy-d-altrose from d-erythrose, as well as 2-deoxy-l-xylose and 2-deoxy-l-ribose from l-glyceraldehyde.