Abstract:Methyltransferases can protect the host genomic DNA from digestion by restriction endonucleases. Based on this property, the classical strategy for preparing restriction endonuclease involves first expressing their paired methyltransferases to protect the host strain, followed by co-expression of the restriction endonuclease, i.e. a 'one-to-one' recombinant expression pattern. In this study, we proposed a novel strategy for generating restriction endonucleases by co-expressing a methyltransferase that could recognize overlapping sequences to protect the host strain. Initially, a methyltransferase recognizing the overlapping sequences was transferred into E. coli ER2566 to protect the host genomic DNA. Subsequently, multiple restriction endonucleases capable of recognizing the overlapping sequences were individually transferred into the host strain to achieve the recombinant expression of restriction endonucleases in a 'one-to-many' recombinant pattern. According to this approach, a series of restriction endonucleases recognizing overlapping sequences (TTAA, AGCT and GCGC) were successfully achieved using methyltransferases M. EsaDix5 I, M. Alu I and M. Hha I, respectively. Additionally, the recombinant expression of two new R. Sac I isozymes, i.e., R.EcoSP4ORF25090P and R.Gma5ORF28P, was also successfully achieved through methyltransferase M. Alu I. This approach simplifies the labor-intensive screening process for host strains resistant to restriction endonuclease digestion, while providing the capability for large-scale preparation of restriction endonucleases. Furthermore, this method can be utilized for the discovery of new isoenzymes or isoschizomers in other microorganisms.
