Structure-guided design and rapid cell-free screening of a superior restriction endonuclease DpnI mutant to enhance the specific activity and efficiency of site-directed mutagenesis

Restriction endonuclease DpnI is a critical tool for eliminating parental templates in site-directed mutagenesis (SDM); however, the wild-type enzyme exhibits low efficiency at low substrate concentrations. In this study, we introduced a lysine mutation into the C-terminal winged helix (wH) domain of DpnI via structure-guided design to enhance electrostatic interactions and established a rapid screening platform using rolling circle amplification coupled with cell-free protein synthesis (CFPS). Results showed that the Q235K variant achieved a specific activity of 640 000 U/mg, 4-fold higher than the wild type, and increased the positive mutation rate in SDM from 82.7% to 92.3% while significantly reducing background plasmid residue. This study not only yielded a high-performance tool enzyme but also demonstrated the high efficiency of CFPS for rapid iteration and optimization in enzyme engineering.