A new high-throughput method for screening 2-deoxyribose-5-phosphate aldolase variants with a higher activity toward aldol reaction of unnatural aldehydes was established for the first time by coupling with an aldehyde dehydrogenase LeADH. The error-prone PCR and site-directed saturation mutagenesis libraries of aldolase LbDERA were constructed and screened using the high-throughput method. Two improved variants, LbDERA(T29L) and LbDERA(F163Y), were identified and combined, giving a double mutant LbDERA(T29L/1F163Y) which showed 7-fold higher activity than the native enzyme. The crystal structure of LbDERA(T29L/1F163Y) obtained by X-ray diffraction with 1.77 angstrom resolution revealed the structural changes responsible for the significant activity improvement. (C) 2016 Elsevier Inc. All rights reserved.