Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.Surface display of heterologous proteins or peptides on bacteria is potentially important in several areas of biotechnology, including development of live vaccine delivery systems, diagnostics, whole-cell absorbents, and novel biocatalysts (11). Lactic acid bacteria (LAB) have the status of being generally recognized as safe (GRAS), making them certainly more useful in food and medical applications than other bacterial species. The development of cell surface display systems for LAB has recently become one of the most active research areas. Most of the cell surface display systems for LAB reported thus far have made use of the C terminus of a cell wall-anchoring protein via an LPXTG motif (8, 12, 19, 24). This anchoring mechanism requires processing by a sortase for covalent anchoring of the protein to the cell wall peptidoglycan (15). Various anchoring proteins, such as membrane-spanning protein PgsA (16) and S-layer protein (3), have also been exploited for surface display. However, heterologous proteins have been anchored to the producer cells, and the use of genetically modified organisms is less desirable or at least still being debated. Surface display of heterologous proteins on genetically unmodified Gram-positive bacteria has been successfully carried out using the peptidoglycan binding lysin motif (LysM) domain of the major autolysin AcmA of Lactococcus lactis (1, 2, 4, 18, 28).LysM was first discovered in the lysozyme of Bacillus phage φ29 as a C-terminal repeat composed of 44 amino acids separated by 7 amino acids (6). LysM is a common module found in more than 4,000 proteins of both prokaryotes and eukaryotes (6). Many bacterial proteins containing LysM are peptidoglycan hydrolases, such as p60 (20), Sep (26), LytF (31), AcmA (5), and Mur (7). The best-characterized LysM-containing protein is the N-acetylglucosaminidase AcmA of L. lactis subsp. cremoris MG1363. AcmA is the major autolysin and is required for cell separation and cell lysis during the stationary phase of L. lactis (5). It contains three domains: the N-terminal signal peptide, an active domain, and a C-terminal peptidoglycan anchor (cA) which consists of three LysM repeats (22). Several functional proteins, including malaria parasite surface antigen, β-lactamase, α-amylase, and viral capsid proteins, have been noncovalently bound to cell walls of AcmA-producing and non-AcmA-producing L. lactis as well as several other Gram-positive bacteria via cA (4, 17, 18, 23, 25).Endolysins from bacteriophages are cell wall hydrolases involved in cell lysis to release the progeny particles from the host cells (9, 30). Most endolysins lack a signal peptide and are translocated across the membrane by the aid of the holin protein. This protein typically contains an N-terminal catalytic domain and a C-terminal cell wall binding domain (33). The endolysins Ply118 and Ply500 of a Listeria monocytogenes phage share a unique C-terminal cell wall binding domain which establishes specific recognition of and high-affinity binding to bacterial cell wall carbohydrates (13). The temperate bacteriophage φPYB5, isolated from the Lactobacillus fermentum YB5 strain, has a hexagonal head, noncontractile tails, and several fibers and belongs to Bradley''s group B as defined by the International Committee on Taxonomy of Viruses (32). The sequence of the endolysin gene lyb5 from the genome of φPYB5 has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"EF531306","term_id":"145687952","term_text":"EF531306"}}EF531306, and the gene product has been successfully expressed in Escherichia coli and has shown a broad lytic spectrum (30).Here, we generated a fusion of green fluorescent protein (GFP) to the C terminus of Lyb5 (Ly5C) to construct a surface display system for LAB. The GFP was bound to the surfaces of various LAB cells by the aid of Ly5C. Moreover, by using the system constructed, β-galactosidase (β-Gal) was functionally displayed on the surfaces of LAB cells and retained its activity.