Use of lac regulatory elements for gene expression in Lactobacillus casei

The lactose operon, lacTEGF, of Lactobacillus casei ssp. casei ATCC393 [pLZ15 -] is encoding an antiterminator protein (LacT), the elements (LacE and LacF) of the lactose-specific phosphotransferase system (PTS) and a phospho--galactosidase (LacG). The lac operon is repressed by glucose and fructose and is induced by lactose, through the PTS/CcpA signal transduction system and an antiterminator mechanism, respectively. Furthermore, the antiterminator activity of LacT is also negatively modulated possibly by a PTS-mediated phosphorylation event. These strong regulatory mechanisms have been used in this work for the design of expression systems. Hence, Bacillus licheniformis -amylase has been efficiently expressed from pIA5lacamy on lactose grown cells. Furthermore, a food-grade mutant, expressing Lactococcus lactis acetohydroxy acid synthase genes (ilvBN), was obtained with an integrative system, developed using lacG and lacF as homologous sequences for recombination. As a result, ilvBN genes were integrated in tandem between lacG and lacF in the chromosome and were co-ordinately expressed with the genes of the lactose operon.