Activation-induced cytidine deaminase-basedin vivocontinuous evolution system enables rapid protein engineering

Directed evolution is a powerful tool to modify the properties of proteins. However, due to multi-round and stage combinations, directed evolution usually requires time- and labor-intensive manual intervention, which limits the efficiency of protein modification to some extent. Therefore,in vivocontinuous evolution system is highly preferred because it can couple the multiple rounds and steps of direction evolution with the host growth cycle, leading to the advantages of effort-saving and accuracy. However, the existing types of this kind of systems can not meet the booming demand. Herein, this paper describes promotedEscherichia coli-assisted continuous evolution (PEACE) that allows forin vivocontinuous evolution of target genes. This system polymorphisms the target gene by activation-induced cytidine deaminase-T7 RNA polymerase (AID-T7 PNAP) fusion protein, then it couples the enzymatic properties of desired variants with the expression of antitoxins to achieve efficient growth-coupled screen using the toxin-antitoxin system (TAS). In this study, T7 RNAP was finally employed for validation of PEACE system, and its specificity to the promoter was successfully altered. These results demonstrated the feasibility and further application potential of PEACE.