Construction And Characterization Of Food-grade Cell Surface Display System In Lactococcus Lactis

Lactic acid bacteria (LAB) are a group of the food-grade microorganisms that have been regarded as non-pathogenic and non-colonized to human, and exhibiting a variety of significant applications in the field of food and medicine manufacturing. One of these bacteria, the Gram-positive Lactococcus lactis, is an important and distinctive species that has drawn a widen attention of investigator's. ?N-acetylmuraminidase (AcmA) is a major autolysin of L. lactis. It has been widely investigated in its structure and function since 1995. In the present study, the gene encoding the AcmA protein, which designated as acmAas, was amplified by PCR from L. lactis wild-type strain AS1.2829, followed by constructing the chromosome-integrated recombinant engineering strains that harboring the partially deleted acmAas fragment in the homologous region of chromosome, to investigate the impact of the partially-mutated AcmA variant on the growth and cell division conditions of the host strain. Further, using AcmAas as the carrier and endo-beta-1,3-1,4-glucanase (GLS) from Bacillus subtilis wild-type BF7658 as the selective marker and the foreign passenger protein, a food-grade chromosome-integrated engineering bacterium with cell-surface displaying activity, was constructed successfully.The BLAST analysis of several previously published acmA sequences was firstly performed to help the design of the conversed primers, and to amplify the AcmA encoding gene from L. lactis AS1.2829 strain. The resulting amplified fragment was sequenced and identified as the AcmA-encoding gene, which was designated as acmAas. Its encoded protein, AcmAas, was characterized the putative structure and domain organization. The overall amino acid sequence of AcmAas was structurally distinguished as an N-terminal catalytic domain with a 57 aa of signal peptide sequence and a C-terminal cell-wall binding domain with three tandemly aligned lysin motifs (LysM) that constitutes a modular secondary structure of "βααβ", and a further blast analysis revealed a significant sequence similarity subjected to the previously identified N-acetylglucosaminidase of L. lactis subsp. lactis IL1403, suggesting AcmAas could be an N-acetylglucosaminidase of AS1.2829. To investigate if the sole LysM domain is functional to bind to cell-wall, a chromosome-integrated engineered strain of AS1.2829, MB257, was constructed by introducing a chimeric gene of "acmAasC₁-Em-acmAasC₂", which elicited an intracellular dual exchanges between host chromosome and the introduced gene. It showed that MB257 strain exhibited normal daughter-cell segregation and growth pattern, indicated that the sole LysM domain can serve as the functional active domain for cell-wall binding. Moreover, this study also present a potential strategy for developing a cell-surface display system in L. lactis that mediated by a chromosome-integrated acmAas anchor.To construct the food-grade chromosome-integrated recombinant strain with cell surface displaying activity, the GLS protein from B. subtilis BF7658 strain was chosed to investigate the feasiability of the food-grade screening marker for the recombinant L. lactis. The recombinant strain of L. lactis, MB138, was constructed and was tested the activity of non-antibiotic selection. It showed the distinct transparent zones around the colonies that grew on the selective medium containing oat, after colorizing by Congolese dye and decolorizing by NaCl, indicating GLS can served as the non-antibiotic selective marker to be used for construction of the chromosome-integrated recombinant L. lactis strain. On the basis of these results, we constructed the homologous recombination plasmid MB260, for further construction of the chromosome-integrated L. lactis strain MB257. In this system, GLS served as the selective marker and the foreign passenger protein, AcmAas contributed to anchor GLS onto the surface of the target cells. The followed approach to analyze the properties of the recombinant strain, and used for surface display other functional foreign protein is under the way.