| Lactic acid bacteria (LAB) have a long traditional use in food industry. With the development of molecular biology, many genetic tools, such as transformation protocols, gene expression vector systems, integration systems and amplification systems are available and have been used to produce a variety of proteins in L. lactis. In recent years, L. lactis has become emerging producer for the production of antigen, allergens and cytokines, which it delivers to mucousal surface of animal and humans. Generally, the ribosomally synthesized proteins in lactic acid bacteria can be targeted to remain in the cytoplasm or be secreted. The secreted proteins can either be anchored to the cell wall or released to the extracellular medium. The anchoring of protein to the surface of bacteria is potentially important in several areas of biotechnological applications, including the development of live vaccine delivery systems, diagnositics, whole-cell absorbents, and novel biocatalysts. Thus, the aim of this thesis was to develop the surface display systems with two different models of anchor protein and evaluate their potential applications in displaying of functional proteins on the surface of lactic acid bacteria. The main results have been obtained as follows: 1. Isolation of intestinal Lactobacillus and analysis of their surface proteinsThe lactic acid bacteria, identified as the natural commensal of intestinal organisms, has been considered to be an important group of bacteria in maintaining the stability of the gastrointestinal tract, protecting intestinal infections and improving the intestinal health and immunity. The ability of adherence to the intestinal epithelial cell is a prerequisite for the colonization of bacteria and a key for their probiotic activities. In this study, a total of 45 lactobacillar isolates from chicken intestinal origin were screened and. tested for their adhesion to human enterocyte-like HT-29 cells. Two strains, Lactobacillus K2-4-3 and K3-1-3 showed higher adherence to HT-29 cells and were completely covered with a layer surface proteins with an approximate molecular mass of 45 kDa and 56 kDa, respectively. The 16s rDNA gene sequences of K2-4-3 and K3-1-3 showed a maximum level of identity to the Lactobacillus crispatus. To determine the gene encoding the S-layer protein, the oligonucleotide primers designed on the basis of conserved gene sequences of S-layer protein from GenBank. Based on the conserved sequence obtained, the specific primers were designed and employed for ligation-anchored PCRs to obtain the adjacent sequences. As a result, the DNA sequence of S-layer protein, termed slpB of Lb. crispatus K2-4-3, was otained. SlpB consisted of a 1,323 bp open reading frame with a protein of 430 amino acids residues and had about 95% similarity to the surface protein gene of Lb. crispatus. 2. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the S-layer proteinPrevious studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provided an approach for targeting the heterologous proteins on the cell surface of lactic acid bacteria. Multiple sequence alignment revealed that the C-terminal region of Lb. crispatus K2-4-3 SlpB (LcsB) had a high similarity with the cell wall binding domains of Lb. acidophilus SA and Lb. crispatus CbsA, suggesting the LcsB can be used as the anchor protein in development of live oral vaccines and the reactions catalyzed by enzymes immobilized on whole cells. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP),? -galactosidase (Gal) and cellulase (CBD) were fused to the N terminus of LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with non-genetically modified lactic acid bacteria cells, the fused GFP, Gal as well as the.fused CBD were functional display on the cell surface of various lactic acid bacteria tested. And the binding capacity could be improved with SDS pretreated cells. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of GFP-LcsB was inserted into the Lactococcus lactis expression vector pSec.Leiss, the GFP could not be secreted into the medium under the control of the nisA promoter. The SDS-PAGE, Western blot, In-gel fluorescence, immunofluorescence microscopy and the SDS sensitivity assay confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of LcsB anchor. In conclusion, LcsB region can be used as a functional scaffold to target the heterologous proteins on the cell surface of lactic acid bacteria in vitro and vivo, and has also the potential in biotechnological applications. 3. Simultaneous display of two haemagglutinin on the cell surface of lactic acid baceria using the LcsB anchorLcsB-mediated surface display system of lactic acid bacteria was used to test the possibility of simultaneous displaying the haemagglutinin HA of H5N1 and H9N2 Avian influenza virus on the surface of lactic acid bacteria. The h5A and h9A gene were amplified by RT-PCR technique and then cloned into the prokaryotic expression vector pET-gfp-lcsB to replace the gfp gene. The obtained plasmid, named pET-h5A-lcsB and pET-h9A-lcsB, were used to express the recombinant fusion proteins H5A-LcsB and H9A-LcsB, respectively. Binding of antigens were achieved by mixing the Lb. johnsonii with the LcsB fusion protiens, SDS-PAGE and immunofluorescence showed that the H5A-LcsB and H9A-LcsB were successfully displayed on the surface of Lb. johnsonii. The surface displayed H5A-LcsB, H9A-LcsB and H5A-LcsB&H9A-LcsB strains were orally administrated to the 6-8 weeks Balb/c mice and the ELISA was used to detect the specific IgA and IgG in the serum after administration. 4. Characterization of a novel LysM domain from Lactobacillus fermentum bacteriophage endolysin and its use as an anchor to display heterologous proteins on the surface of lactic acid bacteriaThe endolysin Lyb5, from Lb. fermentum temperate bacteriophage cpPYB5, 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 LysM repeated regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, the green fluorescent protein (gfp) was fused to the N terminus of Ly5C and the resulting GFP-Ly5C fusion was expressed in E. coli. After mixed with various cells in vitro, the GFP-Ly5C was successfully displayed on the cell surface of L. lactis, Lb. casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbruekii, Lb. helveticus and Streptococcus thermophilus. In comparison with nonpretreated L. lactis and Lb. casei cells, an increase in fluorescence intensity of chemical pretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride was 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 surface display system in lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C mediated surface display system,?-galactosidase (Gal) from Paenibacillus sp. K1 replacing GFP was functionally displayed on the surface of LAB cells via Ly5C. The success in surface displaying GFP and Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB. 5. Construction a synthetic library of nisin inducible promoters for gene expression in L. lactisMetabolic engineering requires steady-state gene expression and the capacity to precisely tune the level of expression. In theory, inducible promoter should be able to meet these requirements by using different level of inducer. However, it is difficult for accurate tuning of multiple gene expression to different levels. A solution has been the use of a synthetic promoter library that is based on the randomized sequences flanking the consensus-10 and-35 promoter regions and allows for fine-tuning of bacterial gene expression. Nisin promoter can effectively control transcription and high expression level of appropriate target gene. Sequence analysis revealed that two-10 and-35 regions are present in the 400 bp region upstream of the nisA ORF. To determine the function of the first promoter P1 of nisA promoter, the promoter PO (containing two-10 and-35 regions) and P2 (containing one-10 and-35 region) were cloned upstream of gfp gene. The fluorescence of GFP drived by P0 and P2 promoters was measured, respectively. The results showed that only the first promoter P1 influenced the nisin inducible efficiency and had no impact on the strength of nisin promoter. Then, the consensus sequence of P2 promoter was kept constant, and the spacer sequence of-10 region to-35 region were randomized. Construction of the synthetic promoter library were performed in vector pLeiss:gfp which gfp gene were downstream of nis promoter. Then, GFP was expressed by synthetic promoters with the induction of 10 ng/ml nisin.35 inducible promoters and 14 constitutive promoters, which covering 3-5 logs of expression levels in small increments of activity, were selected. The synthetic promoters were tested for the ability to drive?-galactosidase expression and got the same results with GFP expression. It is the first report for combination of nisin inducible expression and synthetic promoter library to obtain a synthetic library of nisin inducible promoters for L. lactis. This library should be applicable to precisely tune gene expression to the optimal extent for metabolic engineering purpose. |
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