Directed Evolution of Restriction Endonuclease BstYI to Achieve Increased Substrate Specificity

Restriction endonucleases have proven to be especially resistant to engineering altered substrate specificity, in part, due to the requirement of a cognate DNA methyltransferase for cellular DNA protection. The thermophilic restriction endonuclease BstYI recognizes and cleaves all hexanucleotide sequences described by 5′-R↓GATCY-3′ (where R=A or G and Y=C or T). The recognition of a degenerate sequence is a relatively common feature of the more than 3000 characterized restriction endonucleases. However, very little is known concerning substrate recognition by such an enzyme. Our objective was to investigate the substrate specificity of BstYI by attempting to increase the specificity to recognition of only AGATCT. By a novel genetic selection/screening process, two BstYI variants were isolated with a preference for AGATCT cleavage. A fundamental element of the selection process is modification of the Escherichia coli host genomic DNA by the BglII N4-cytosine methyltransferase to protect AGATCT sites. The amino acid substitutions resulting in a partial change of specificity were identified and combined into one superior variant designated NN1. BstYI variant NN1 displays a 12-fold preference for cleavage of AGATCT over AGATCC or GGATCT. Moreover, cleavage of the GGATCC sequence is no longer detected. This study provides further evidence that laboratory evolution strategies offer a powerful alternative to structure-guided protein design.