Catalytic Turnover-Based Phage Selection for Engineering the Substrate Specificity of Sfp Phosphopantetheinyl Transferase

A general strategy for the evolution of bond-forming enzymes using yeast display

“…Our results highlight the attractive features of yeast display that offer significant advantages for enzyme evolution, including quality control mechanisms within the secretory pathway that ensure display of properly folded proteins and compatibility with FACS (18). For these reasons, we used yeast as the vehicle for display instead of an M13 phage simultaneously displaying an Sfp peptide substrate and an enzyme library (33). As the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it is in principle compatible with any bond-forming enzyme that can be expressed in yeast, including glycosylated proteins that are likely incompatible with phage and mRNA display, provided that linkage of the substrates to CoA and to the affinity handle is possible and tolerated by the enzyme or its evolved variants.…”

Enzyme‐Catalyzed Substrate Attachment to Phage Surfaces for the Selection of Catalytic Activities

“…[20] We recently developed a method for the codisplay of enzymes and peptide substrates on the phage surface and used this method to evolve the protein post-translational modification (PTM) enzyme Sfp. [21] Sfp is a phosphopantetheinyl transferase that can modify an 11-residue peptide, ybbR, with smallmolecule probes conjugated to coenzyme A (CoA). [22] To select for Sfp mutants that utilize 3'-dephospho CoA (dpCoA) in ybbR modification, we linked the coding sequence for the ybbR peptide to the pIII gene in the genome of M13KO7 helper phage to construct the helper phage "ybbR-M13".…”

“…[22] To select for Sfp mutants that utilize 3'-dephospho CoA (dpCoA) in ybbR modification, we linked the coding sequence for the ybbR peptide to the pIII gene in the genome of M13KO7 helper phage to construct the helper phage "ybbR-M13". [21] ybbR-M13 produces phage particles with the ybbR peptide fused to the N terminus of the pIII capsid protein that is exposed on the phage surface (Scheme 1 G). During phage proScheme 1.…”

“…Using this method, we identified mutants of Sfp that use dpCoA for ybbR modification. [21] We took advantage of the efficient protein-labeling reaction catalyzed by Sfp to prepare substrate-attached phage for the selection of catalytic activities. Sfp has substantial substrate promiscuity and it can covalently attach small molecules of diverse structures including biotin, carbohydrates, peptides, and fluorophores to the ybbR peptide.…”

“…We first confirmed the display of ybbR and BirA on the phage surface. To test the display of the ybbR peptide, N-and C-BirA phage were labeled with a biotin-CoA [21] conjugate catalyzed by Sfp. The labeling reactions were subjected to PAGE.…”