Deciphering the mechanisms of Limosilactobacillus fermentum A51 involved in exopolysaccharides biosynthesis regulated by LuxS/AI-2-QS system

Limosilactobacillus fermentum A51 is a high Exopolysaccharides (EPS) producing strain harboring the S-ribosylhomocysteinase/autoinducer-2-quorum sensing (LuxS/AI-2-QS) system. However, the regulatory mechanism of EPS synthesis by this system remains unclear. In this study, we combined biochemical, proteomic, and parallel reaction monitoring (PRM) analyses to elucidate the role of the LuxS/AI-2-QS system in EPS biosynthesis. Biochemical results indicated that 200 μmol/L furanone inhibitor could downregulate luxS and eps gene expression, reducing the AI-2 activity and EPS yield (decreased from 459.87 mg/L to 439.26 mg/L) and monosaccharide yield (P < 0.05), thereby blocking the LuxS/AI-2-QS system and EPS biosynthesis of L. fermentum A51. Thus, the LuxS/AI-2-QS system positively regulates EPS synthesis. Furthermore, tandem mass tags (TMT)-based quantitative proteomics identified 512 differentially expressed proteins (DEPs) between normal and furanone-inhibited groups. Among them, 32 downregulated DEPs in the inhibited-group, and they were involved in the LuxS/AI-2-QS system and Wzx/Wzy-dependent pathway. PRM validation confirmed twelve key proteins, including AI-2 synthesis protein LuxS, AI-2 export transporter (AI-2E), and the EPS biosynthesis-related proteins glycosyl transferase, glucokinase, flippase (Wzx), and polymerase (Wzy). Overall, this study provides novel insights into the LuxS/AI-2-QS system regulating EPS biosynthesis of LAB by mediating the Wzx/Wzy pathway, which might promote development of new approaches for EPS biosynthesis based on QS target.