High-level gene expression in Lactobacillus plantarum using a pheromone-regulated bacteriocin promoter

Abstract

Aims:1 To use promoters and regulatory genes involved in the production of the bacteriocin sakacin P to obtain high-level regulated gene expression in Lactobacillus plantarum. Methods and Results: In a plasmid containing all three operons naturally involved in sakacin P production, the genes encoding sakacin P and its immunity protein were replaced by the aminopeptidase N gene from Lactococcus lactis (pepN) or the b-glucuronidase gene from Escherichia coli (gusA). The new genes were precisely fused to the start codon of the sakacin P gene and the stop codon of the immunity gene. This set-up permitted regulated (external pheromone controlled) overexpression of both reporter genes in L. plantarum NC8. For PepN, production levels amounted to as much as 40% of total cellular protein.Conclusions: Promoters and regulatory genes involved in production of sakacin P are suitable for establishing inducible high-level gene expression in L. plantarum. Significance and Impact of the Study: This study describes a system for controllable gene expression in lactobacilli, giving some of the highest expression levels reported so far in this genus.

The Major Autolysin Acm2 from Lactobacillus plantarum Undergoes Cytoplasmic O-Glycosylation

“…Fresh overnight cultures of L. plantarum WCFS1 and its derivatives were diluted in fresh MRS and induced at an optical density at 600 nm (OD 600 ) of 0.1 by adding 25 g/ml peptide pheromone, as previously described (25). After 6 h of growth at 37°C, the cells were harvested by centrifugation at 3,000 ϫ g for 10 min at 4°C.…”

Microbial production of prebiotic oligosaccharides

“…Different approaches to engineering β-galactosidases and glycosidases were quite successful in producing improved enzymes with high transglycosylation activity that could be effi ciently used in the synthesis of oligosaccharides (Hancock et al, 2006;Feng et al, 2005). Recently, a new food-grade host/vector system for L. plantarum based on the pSIP expression vectors (Sørvig et al, 2003(Sørvig et al, , 2005Mathiesen et al, 2004aMathiesen et al, , 2004b was constructed using the homologous alanine racemase gene (alr) as selection marker . The vectors were applied for intracellular overexpression of the β-galactosidase genes from various Lactobacillus spp.…”

Characterization of a Cryptic Plasmid pD403 from Lactobacillus plantarum and Construction of Shuttle Vectors Based on its Replicon

“…Although evidence of these beneficial effects is rapidly accumulating, the basic knowledge relating to the mechanisms by which Lactobacillus contributes to other bacteria or host cells is still poor [4]. Such knowledge has aroused an increased interest in their genetics, and a variety of genetic tools have been developed in recent years [5,6].…”