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Applied and Environmental Microbiology, June 2000, p. 2599-2604, Vol. 66, No. 6
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 (
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
-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
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
-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.
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