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 food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (beta-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 beta-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific 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 beta-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.
Martín
M C
MC
Departamento de Biología Funcional, Area Microbiología and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain.
Alonso
J C
JC
Suárez
J E
JE
Alvarez
M A
MA
D000818:Animals; D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004268:DNA-Binding Proteins; D005285:Fermentation; D005516:Food Microbiology; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000821:virology; D008892:Milk / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D045522:Recombinases; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D045522:Recombinases; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1