Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

Abstract

The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (␤-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding ␤-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by sitespecific recombination, integrated as a single copy in an orientation-and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the ␤-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.

Lactococcus lactis Lytic Bacteriophages of the P335 Group Are Inhibited by Overexpression of a Truncated CI Repressor

“…An A2 repressor gene integrated into the chromosome of Lactobacillus casei rendered the strain completely resistant to phage A2 (2,29). Background expression of the phage adh repressor in Lactobacillus gasseri also conveyed complete resistance to phage adh (13).…”

Reporter systems forin vivotracking of lactic acid bacteria in animal model studies

“…28,82 Plasmid reporter gene expression levels are usually higher than those of some chromosomally integrated genes. 83 Translation efficiency of a protein in a heterologous host organism can be significantly increased by modifying the codon usage frequency, thus codon optimization. 84 To enhance the translational efficiency of a reporter gene, the gene can be chemically synthesized with an optimized codon usage for expression in specific LAB.…”

Genetically Engineered Lactobacilli for Technological and Functional Food Applications

“…A subsequent temperature shift selects for loss of the helper plasmid and integration of the pOWV01-derived vector. In addition, there are other mutagenesis systems as that of the Cre-lox-based system used in L. plantarum (Lambert et al, 149 2007) and site-specific integrative vectors based on prophage fragments (Martin et al, 2000). Other important genetic tool used to study chromosomal genes and their regulation in lactobacilli is random transposon mutagenesis.…”