Construction of a Bile-responsive Expression System in Lactobacillus plantarum

Abstract

This study aimed to develop a bile-responsive expression system for lactobacilli. The promoters of four genes, encoding phosphoenolpyruvate-dependent sugar phosphotransferase (mannose-specific), L-lactate dehydrogenase (LDH), HPr kinase, and D-alanine-D-alanine ligase, respectively, which were highly expressed by bile addition in <i>Lactobacillus johnsonii</i> PF01, were chosen. Each promoter was amplified by polymerase chain reaction and fused upstream of the β-glucuronidase gene as a reporter, respectively. Then, these constructs were cloned into <i>E. coli</i>-<i>Lactobacillus</i> shuttle vector pULP2, which was generated by the fusion of pUC19 with the <i>L. plantarum</i> plasmid pLP27. Finally, the constructed vectors were introduced into <i>L. plantarum</i> for a promoter activity assay. The LDH promoter showed the highest activity and its activity increased 1.8-fold by bile addition. The constructed vector maintained in <i>L. plantarum</i> until 80 generations without selection pressure. A bile-responsive expression vector, pULP3-P_LDH, for <i>Lactobacillus</i> spp. can be an effective tool for the bile-inducible expression of bioactive proteins in intestine after intake in the form of fermented dairy foods.