Complementary enhancement of osmotic tolerance in Ligilactobacillus salivarius AR612 by exogenous purine nucleobases and gene engineering.

High-density fermentation and high-sugar environments place substantial osmotic stress on the probiotic strain Ligilactobacillus salivarius, causing its viability to be insufficient for food industrial applications, biological preservation, and probiotic formulation development. We investigated the efficacy of exogenous nucleobases and genetic engineering for enhancing the hyperosmotic tolerance of L. salivarius AR612. A thorough investigation using physiological assessments, molecular cloning, and metabolomics demonstrated that supplementation with purine nucleobases, particularly adenine, enhances growth performance in high-glucose and high-salt environments while preserving cellular integrity. The overexpression of genes related to sugar transport (e.g., scrA, gene0084, rbsB, lacS, artQ, manZ), heat shock responses (groES, gene1369), and energy metabolism (ndh) was found to enhance hyperosmotic tolerance, whereas downregulation of these genes exacerbated sensitivity to stress. Metabolomic analysis identified common regulatory pathways underlying the protective effects of both purine nucleobases supplementation and gene overexpression, including nucleotide metabolism, the pentose phosphate pathway, and amino acid metabolism. Fermentation trials conducted in high-glucose medium and jujube juice confirmed the robustness of the engineered strains. These results demonstrate the complementary roles of exogenous nucleobases and functional genes in alleviating osmotic stress and provide a strategic framework for enhancing probiotic efficacy in industrial and food applications.