Catalytic turnover-based phage selection for engineering the substrate specificity of Sfp phosphopantetheinyl transferase.

We report a high-throughput phage selection method to identify mutants of Sfp phosphopantetheinyl transferase with altered substrate specificities from a large library of the Sfp enzyme. In this method, Sfp and its peptide substrates are co-displayed on the M13 phage surface as fusions to the phage capsid protein pIII. Phage-displayed Sfp mutants that are active with biotin-conjugated coenzyme A (CoA) analogues would covalently transfer biotin to the peptide substrates anchored on the same phage particle. Affinity selection for biotin-labeled phages would enrich Sfp mutants that recognize CoA analogues for carrier protein modification. We used this method to successfully change the substrate specificity of Sfp and identified mutant enzymes with more than 300-fold increase in catalytic efficiency with 3'-dephospho CoA as the substrate. The method we developed in this study provides a useful platform to display enzymes and their peptide substrates on the phage surface and directly couples phage selection with enzyme catalysis. We envision this method to be applied to engineering the catalytic activities of other protein posttranslational modification enzymes.