Food-grade expression system of Lactobacillus plantarum using B-galactosidase small subunit as selection marker and lactose as screening condition

Lactobacillus plantarum, one of the most important and widely studied lactic acid bacteria, has been used in many fields. In recent years, the construction and application of food-grade expression systems in L. plantarum has shown significant advantages in the fields of food and medicine. In this study, a food-grade expression system of L. plantarum with B-galactosidase as a screening marker was constructed by exploring the complementary properties of the gene lacLM of B-galactosidase in L. plantarum. Firstly, L. plantarum knockout strains AlacM and AlacAlacM were constructed, and it was determined that the knockout strain AlacAlacM was endowed with the phenotype of being unable to metabolize lactose. The knockout strain AlacAlacM was selected as the food-grade host for the food-grade expression system. The lacM gene was further expressed by plasmid pMG36e and transformed into L. plantarum AlacAlacM. The complementary strain restored lactose utilization ability, and its biomass reached 87.3% of that of L. plantarum LP01. Based on this phenomenon, a food-grade vector pMG36m with lacM gene as a selection marker was obtained by removing the antibiotic resistance gene. The genetic stability test showed that the plasmid existence rate was still close to 100% after 98 generations of L. plantarum AlacAlacM/pMG36m. Subsequently, an expression vector pMG36m-gfp was constructed with green fluorescent protein gene (gfp) as the reporter gene. These results indicated that a novel food-grade expression system of L. plantarum with lacM gene as a selection marker and lactose as a screening condition was successfully estab-lished, laying a foundation for the application of L. plantarum.