Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination

The construction of a delivery and clearing system for the generation of fo od-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (beta-recombinase) and their respec tive target sites (attP-attB and sin, respectively) is reported. The delive ry system contains a heterologous replication origin and antibiotic resista nce markers surrounded by two directly oriented six sites, a multiple cloni ng site where passenger DNA could be inserted (e.g., the cI gene of bacteri ophage A2), the int gene, and the attP site of phage A2. The clearing syste m provides a plasmid-borne gene encoding beta-recombinase, The nonreplicati ve vector-borne delivery system was transformed into Lactobacillus casei AT CC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the aftB site present in the genome of the host strain. The transfer of the clearing system into this st rain, with the subsequent expression of the beta-recombinase, led to site-s pecific DNA resolution of the non-food-grade DNA. These methods were valida ted by the construction of a stable food-grade L. casei ATCC 393-derived st rain completely immune to phage A2 infection during milk fermentation.