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Applied and Environmental Microbiology, June 2000, p. 2599-2604, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

M. Cruz Martín,1,2 Juan C. Alonso,3 Juan E. Suárez,1,2 and Miguel A. Alvarez2,*

Departamento de Biología Funcional, Area Microbiología and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo,1 Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid,3 and Instituto de Productos Lácteos de Asturias (CSIC), 33300 Villaviciosa, Asturias,2 Spain

Received 24 January 2000/Accepted 21 March 2000

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.

* Corresponding author. Mailing address: Instituto de Productos Lácteos de Asturias (CSIC), Ctra. de Infiesto s/n, Apartado de Correos 85, 33300 Villaviciosa, Asturias, Spain. Phone: (34) 985892131. Fax: (34) 985892233. E-mail: maag{at}ipla.csic.es.

Applied and Environmental Microbiology, June 2000, p. 2599-2604, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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