High-throughput screening methods for selecting l-threonine aldolases with improved activity

Abstract More and more, aldolases are being recognized as useful catalysts that carry out the reversible addition of a ketone donor to an aldehyde acceptor in achieving high stereoselectivity. Threonine aldolases catalyze the synthesis of variable β-hydroxy-α-amino acids, which are important structural units of various antibiotics and immunosuppressants. However, the enzymatic properties need to be improved to support a broader application to synthetic chemistry. Although directed-evolution is a powerful tool for improving enzymatic properties, the successful outcome depends on the efficiency of screening systems. We designed and proposed two high-throughput screening schemes for selecting l -threonine aldolase mutants with improved properties. These schemes utilized the toxicity of aldehyde, which acts as an acceptor in the aldol condensation. In these schemes, the following occurs: (1) the higher l -threonine aldolase activity reduces the toxic effect of aldehyde, which leads to the survival of the corresponding clone (the positive-selection scheme), and (2) the higher l -threonine aldolase activity produces more toxic aldehyde, which causes the death of the corresponding clone (the negative-selection scheme). According to the positive-selection scheme, we successfully selected l -threonine aldolase mutants with higher activities than the wild-type, from a randomly generated LTA library.