Development of a LytE-based high-density surface display system in Bacillus subtilis

Summary The three N‐terminal, tandemly arranged LysM motifs from a Bacillus subtilis cell wall hydrolase, LytE, formed a cell wall‐binding module. This module, designated CWBMLytE, was demonstrated to have tight cell wall‐binding capability and could recognize two classes of cell wall binding sites with fivefold difference in affinity. The lower‐affinity sites were approximately three times more abundant. Fusion proteins with β‐lactamase attached to either the N‐ or C‐terminal end of CWBMLytE showed lower cell wall‐binding affinity. The number of the wall‐bound fusion proteins was less than that of CWBMLytE. These effects were less dramatic with CWBMLytE at the N‐terminal end of the fusion. Both CWBMLytE and β‐lactamase were essentially functional whether they were at the N‐ or C‐terminal end of the fusion. In the optimal case, 1.2 × 107 molecules could be displayed per cell. As cells overproducing CWBMLytE and its fusions formed filamentous cells (with an average of nine individual cells per filamentous cell), 1.1 × 108β‐lactamase molecules could be displayed per filamentous cell. Overproduced CWBMLytE and its fusions were distributed on the entire cell surface. Surface exposure and accessibility of these proteins were confirmed by immunofluorescence microscopy.