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, ␤-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.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

“…The RepA proteins from pSF118-20 and pSF118-44 (LSL_1965 [repA 20 ] and LSL_2000 [repA 44 ], respectively) are 71% identical to each other and are 72% similar to the Rep protein of lactococcal plasmid pCI2000, which is predicted to replicate via a theta replication mechanism (31).…”

“…Distribution of related plasmids with theta-type replicons in L. salivarius strains. The detection of pSF118-20 repA (repA 20 )-and pSF118-44 repA (repA 44 )-related plasmids in 27 strains of L. salivarius was performed by a combination of PFGE and Southern hybridization. Nuclease S1-treated genomic DNA samples of L. salivarius strains were resolved by PFGE (Fig.…”

“…Probes based on repA 20 and repA 44 were generated by PCR and hybridized to membrane blotted with genomic DNA Fig. 2A and C, as repA 20 and repA 44 are 73% identical in nucleotide sequences. Hybridization signals in Fig.…”

“…Therefore, 52% of the tested 27 L. salivarius strains harbor plasmids ranging from 10 to 70 kb, which are all repA 20 -or repA 44 -related plasmids. Most of the plasmids from the L. salivarius strains are both repA 20 and repA 44 related due to their high sequence-relatedness identity, except for the small plasmids from NCIMB8818 (10 kb) and CCUG47826 (15 kb) that are repA 20 related and the plasmids from CCUG47171 that are repA 44 related. Curing of pSF118-20.…”

“…The L. salivarius strains used in this study are listed in Table 2. E. coli Top10 was used as an intermediate host for the pCI341 and pEM constructs and was grown in Luria-Bertani broth (20) with aeration at 37°C. L. salivarius, L. plantarum, and L. fermentum were grown under microaerobic conditions (5% CO 2 ) in de ManRogosa-Sharpe (MRS) medium (Oxoid Ltd., United Kingdom) at 37°C, except when simultaneously detecting bioluminescence and growth.…”