**3. Applications of surface engineering of LAB**

432 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

cultures (Turner et al., 2004).

*2.2.3. WxL anchors* 

*2.2.4. Other anchors* 

1995). The C-terminus of endolysin Lyb5 of *Lactobacillus fermentum* bacteriophage øPYB5 (Ly5C) contains three LysMs. Each of LysMs is composed of 41 amino acids and they are separated by intervening sequences varying in length and composition. Ly5C fused to GFP was expressed in *E. coli*. After mixing the fusion protein with various cells *in vitro*, GFP was successfully displayed on the surfaces of *L. lactis, L. casei*, *L. brevis*, *L. plantarum*, *L. fermentum*, *L. delbrueckii*, *L. helveticus*, and *S. thermophilus* cells. Increases in the fluorescence intensities of TCA treated *L. lactis* and *L. casei* cells compared to those of nontreated cells showed that the cell wall peptidoglycan was the cell surface binding target of Ly5C. Concentration of sodium chloride and pH influenced the binding capacity of the fusion protein, and optimal conditions of these factors were determined empirically in order to obtain high fluorescence intensities of *L. lactis* and *L. casei* cells (Hu et al., 2010). N-terminus of putative muropeptidase (MurO) of *L. plantarum* also contains two LysMs composed of about 43 amino acids separated by 22 amino acid residue sequences. The LysM domain fused to GFP was expressed in *E. coli* and it was able to bind to the cell surface of *L. plantarum* after being mixed with the cells (Xu et al., 2011). Examination of supernatant fractions from broth cultures of *L. fermentum*, revealed the presence of a 27-kDa protein termed Sep. The N-terminus of Sep contains a LysM. Sep fused N-terminally to a six histidine epitope was expressed in *L. fermentum*, *Lactobacillus rhamnosus,* and *L. lactis*. The protein was found associated with the surface of the expression hosts. However, it was largely present in the supernatant of the cell

The C-terminal cell wall binding domain designated WxL was first identified in proteins of *Lactobacillus* and other LAB based on *in silico* analysis (Kleerebezem et al., 2010). WxL domain contains a WxL motif followed by a proximal well conserved YXXX(L/I/V)TWXLXXXP motif. This domain was found in gene clusters that also encode additional extracellular proteins with C-terminal membrane anchors and LPxTG motif containing anchors, suggesting that they form an extracellular protein complex (Siezen et al., 2006). The C-terminal WxL domains identified in two proteins of *Enterococcus faecalis* were fused at their N-termini to an export reporter enzyme (nuclease of *S. aureus)* and a secretion signal peptide. The fusion proteins expressed in *E. faecalis* were detected in both cell wall and supernatant fractions of the recombinant enterococci. Removal of the WxL domains from the fusion proteins nearly eliminated them in the cell wall. Treatment of the cell wall fractions with SDS disrupted binding of the fusion proteins to these fractions. These results indicated that the fusion proteins had noncovalent interactions with the cell wall of *E. faecalis*. The fusion proteins were able to attach to the cell surface of *E. faecalis* and *L. johnsonii*

Basic surface protein A (BspA) is a surface located protein of *L. fermentum* BR11. Sequence comparisons have been shown that BspA is a member of family III of the solute binding

when they were added exogenously (Brinster et al., 2007).

Research in the field of surface engineering of LAB has mainly been focused on the construction of vaccine delivery vehicles but other interesting applications have also been reported. In this section, we will describe different areas of biotechnology in which surface display of heterologous proteins on LAB have been investigated.
