**1. Introduction**

Anchoring of proteins to the cell surface is a common theme in nature and the processes governed by different surface proteins are bases of many biological phenomena, such as cellcell recognition, signal transduction, adherence, colonization, and immunoreactions (Westerlund & Korohonen, 1993). The utilization of cellular surface anchoring systems for the display of heterologous proteins on the surface of microbial cells has been developed into an active research area that holds a great promise for a variety of biotechnological applications including the production of whole cell biocatalysts, microbial adsorbents, live vaccines, antibody fragments, and screening of novel proteins (Hansson et al., 2001; Kondo& Ueda, 2004; Lee et al., 2003). Generally construction of these systems is accomplished by the expression of heterologous peptides or proteins as fusions with anchoring domains, which are able to attach to the cell surface (Fig 1.). Anchoring domains are usually cell surface proteins or their fragments. Depending on the characteristics of target and anchor proteins, N-terminal fusion, C-terminal fusion or sandwich fusion strategy can be considered (Lee et al., 2003).

**Figure 1.** Schematic representation of a microbial surface display system

© 2013 Tarahomjoo, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

LAB are gram positive, non-spore forming, fastidious, acid tolerant, and strictly fermentative that secret lactic acid as the major end product of sugar fermentations (Axelsson, 1998). LAB are naturally present in media rich in organic nutrients such as food products and digestive tracts. They are a genetically diverse group of bacteria with GC contents varying from 34 to 53%, including rod shaped bacteria such as lactobacilli and also cocci such as lactococci, enterococci, pediococci, and leuconostoc (Stiles & Holzapfel, 1997). Since time immemorial, LAB have been used for the fermentation and preservation of food products, particularly dairy products, fermented meats, and vegetables. Consequently, several strains of LAB have a long record of safe association with humans and human foodstuffs (Mckay & Baldwin, 1990). The display of proteins on the surface of LAB cells can broaden or improve applications of these bacteria. In this chapter, we intend to describe cell surface anchoring domains used in LAB surface display systems. Then applications of surface engineered LAB are depicted and key factors affecting their performances are highlighted. Moreover, we explained comprehensively a novel application of the protein display in LAB, which is potentially useful for enhancement of the delivery of viable LAB to the gastrointestinal tract (GIT).

Exploring Surface Display Technology

for Enhancement of Delivering Viable Lactic Acid Bacteria to Gastrointestinal Tract 429

The most widely used surface display systems of LAB are based on cell wall anchored proteins that contain an LPXTG motif. These proteins are first synthesized as a preprotein containing an N-terminal signal peptide and a C-terminal cell wall anchor domain. The anchor domain starts at its C-terminus with a short tail of positively charged residues (five to seven amino acids) that remain in the cytoplasm. Upstream of the cytoplasmic domain, a stretch of approximately 30 hydrophobic amino acids is preceded by the highly conserved pentapeptide LPXTG. The charged tail and hydrophobic domain are thought to function as a temporary stop to position the LPXTG motif for proteolytic cleavage. Correct positioning results in cleavage between the threonine and glycine residues followed by amid linkage of the threonine residue to the peptide crossbridge in the peptidoglycan of cell wall, by the action of a sortase (Navarre & Schneewind, 1994). The amino acid composition of the peptide crossbridge varies among the different LAB species and is flexible with respect to the sorting reaction (Strauss et al., 1998; Ton-That et al., 1998). The anchor domain is preceded by a wall associated region of about 50 to 125 residues and is characterized by a high percentage of proline/glycine and/or threonine/serine residues (Fischetti et al., 1990). In surface display genetic constructs, secretion signal peptides are fused to the N-termini of target proteins to transport them to the outside of cell and the LPXTG motif containing anchors are fused to the C-termini of target proteins in order to attach them to the cell surface. The cell surface associated proteinases of *Lactococcus lactis* and *Lactobacilli casei*  (PrtPs), M6 protein of *Streptococcus pyogenes*, and Protein A of *Staphylococcus aureus* (SpA) are among LPXTG type anchors, which have mainly been used for the construction of surface display systems in LAB (Maassen et al., 1999; Norton et al., 1996; Piard et al., 1997; Pouwels

Slight deviations from the LPXTG consensus sequence is found in some LAB. Recently Kleerebezem (2003) found that sortase substrates of *Lactobacillus plantarum* contain LPQTXE motifs instead of the LPXTG motifs. In addition, in the carboxy end of cell surface proteinase of *Lactobacillus delbrueckii* (PrtB), a degenerated LPKKT motif is surrounded by two imperfect repeats of 59 residues, which are lysine rich. However, downstream of the LPKKT motif, there is no hydrophobic domain and no charged tail at the extreme C-terminus. It was shown that the C-terminal region of PrtB was able to attach to the cell wall of *L. lactis* and the capacity of attachment was drastically reduced by absence of the duplicated sequences. The high content of total positive charges in the anchoring region of PrtB suggests interactions of the anchor with the negatively charged teichoic acids of the cell wall. The mechanism of PrtB attachment to the cell wall probably implicates electrostatic forces

Lipoproteins are lipid modified proteins produced as secretory precursors with a signal peptide linked to their amino termini. The C-terminal region of their signal peptide contains

**2.1. Covalent anchors** 

*2.1.1. LPXTG motif containing anchors* 

et al., 1996; Steidler et al., 1998).

(Germond et al., 2003).

*2.1.2. Lipoprotein anchors* 
