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Applied and Environmental Microbiology, November 2000, p. 4822-4828, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Integrative Food-Grade Expression System Based on the Lactose Regulon of Lactobacillus casei

María José Gosalbes, Carlos David Esteban, José Luis Galán, and Gaspar Pérez-Martínez*

Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, 46100-Burjassot, Valencia, Spain

Received 3 July 2000/Accepted 6 September 2000

The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3' end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, beta -glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.

* Corresponding author. Mailing address: Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de los Alimentos (C.S.I.C.), Polígono de la Coma s/n, Apartado de correos (P.O. Box) 73, 46100-Burjassot, Valencia, Spain. Phone: 34 96 3900022. Fax: 34 96 3636301. E-mail: gaspar.perez{at}iata.csic.es.

Applied and Environmental Microbiology, November 2000, p. 4822-4828, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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