Integrative food-grade expression system based on the lactose regulon of Lactobacillus casei

Abstract

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.

FIG. 1

Restriction maps of integrative vectors. (A) Integrative vector pIlac. Erm and Ap are erythromycin and ampicillin resistance genes: ori represents the E. coli replicon. lacG and lacF genes encode P-β-Gal and EIIA^lac of the lac operon. The 45 bp of intergenic region are shown with the newly created restriction sites and an RBS. (B) Vector pIlacgus. The gusA gene was cloned into PstI/EcoRI pIlac. Vector pIlacilv was constructed by cloning ilvBN genes into NdeI/EcoRI pIlac.

FIG. 2

Southern blot of total DNA digested with BglII/PstI from L. casei CECT 5276 (lane 1), an integrant of pIlacilv (lane 2), and L. casei CECT 5291 (food-grade integrant) (lane 3). The probes used were Plac (A), Pilv (B), and Perm (C). M represents digoxigenin-labeled λ phage DNA digested with HindIII as a molecular size marker.

FIG. 3

End metabolite production (acetaldehyde, ethanol, diacetyl, acetoin, ALA, acetone, 1, butanol, and lactic acid) by the wild type (1) and ilvBN integrant (2) from cells grown on glucose plus lactose (black), lactose (white), and ribose (grey). The values are from at least three independent experiments, and the coefficient of variation for each mean was less than 10%.