Protein engineering of aldolase LbDERA for enhanced activity toward real substrates with a high-throughput screening method coupled with an aldehyde dehydrogenase

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/F163Y which showed 7-fold higher activity than the native enzyme. The crystal structure of LbDERA_T29L/163Y obtained by X-ray diffraction with 1.77 Å resolution revealed the structural changes responsible for the significant activity improvement. - Highlights: • A high-throughput method for screening aldolase variants was established. • A variant LbDERA_T29L/F163Y with 7-fold improved activity was identified. • The crystal structure reveals structure changes responsible for activity improvement.