Three threonine aldolases (TAs) were cloned and overexpressed in Escherichia colt (Aeromonas jandaei L-allothreonine aldolase, E. coli L-threonine aldolase and Thermotoga maritima L-alto-threonine aldolase). A Design of Experiments strategy was used to identify optimal reaction conditions for each enzyme. These conditions were used to characterize the substrate- and stereoselectivity of each TA toward a panel of aldehyde acceptors. In general, the A. jandaei TA performed best, and six representative conversions were scaled up 10-fold in order to develop downstream steps for product isolation. One key improvement was to treat the crude reaction product with Bacillus subtilis glycine oxidase, which eliminated residual starting material and significantly simplified product isolation. NMR studies were used to identify the major and minor diastereomers from the preparative scale reactions and the absolute configurations for three representative cases.