Development of strong lactose/galactose-inducible expression system for Lactobacillus plantarum by optimizing promoter

•Two lactose/galactose-inducible promoters were identified in Lb. plantarum WCFS1.•Hybridized promoter strategy was employed to improve the strength of the promoter.•Series strong inducible promoters were obtained by optimizing promoter elements.•Optimized promoters can be used to express heterologous proteins in Lb. plantarum. The lactose-inducible expression system was widely used in bacteria due to the safety of lactose. However, the strength of its promoter was limited in lactic acid bacteria. Here, the β-galactosidase activity in Lactobacillus plantarum WCFS1 was detected at the presence of lactose and galactose. Sequence analysis showed that two β-galactosidase encoding genes lacA and lacLM were identified in the genome of Lb. plantarum WCFS1. Using green fluorescent protein as a reporter, we found that the promoter PlacA of lacA was lactose-induced, while promoter PlacLM of lacLM was lactose/galactose-induced. Furthermore, by optimizing the sequences of –35, –10 regions and ribosome binding sites of these two promoters, the maximum strength of the promoter PlacA derivative increased 10.4-fold by lactose induction, while PlacLM derivative increased 12.7-fold by lactose induction and 9.0-fold by galactose induction compared with their original promoters, respectively. When the optimized promoters were used to drive the β-galactosidase gene from Streptococcus thermophilus CGMCC 7.179, the highest β-galactosidase activity was 45.72 ± 0.44 U/mL under PlacLM-35-10 control. The strength of these promoters was also confirmed by western-blot analysis. Thereby, this study provided a set of novel lactose/galactose-inducible promoters with high expression efficiency and thus expanded the toolbox to express recombinant protein in Lb. plantarum.