Metabolomics analysis to explore the potential mechanism of MntR and SecA in Lacticaseibacillus rhamnosus Gr18 biofilm formation under Mn2+ deficiency

Biofilm formation promotes Lactic acid bacteria (LAB) encapsulation in extracellular matrix, which increases viability during processing. Previous research has shown that Mn 2+ deficiency could promote Lacticaseibacillus rhamnosus Gr18 biofilm formation, with MntR and SecA serving functions. This study elucidated potential mechanism of MntR and SecA during adhesion and maturation stage of L. rhamnosus Gr18 biofilm formation by using gene editing and untargeted metabolomics. Results showed that after mntR knockout and secA overexpression, biofilm biomass has been reduced by 39.16 % and 22.89 %. The mntR knockout decreased extracellular polysaccharide and protein level. MntR influenced carbon/nitrogen balance during adhesion stage, enhanced biofilm integrity via cysteine metabolism during maturation stage. The secA overexpression reduced extracellular lipid and DNA content. SecA remodeled matrix composition via amino acid metabolism during adhesion stage, combined AI-2 and c-di-GMP to refine biofilm structure during maturation stage. MntR and SecA simultaneously affected purine and pyrimidine metabolism to promote extracellular matrix synthesis throughout biofilm formation. Fructose, thymine, and xanthine were identified as key metabolites during formation. These findings illustrated mechanism of promoting biofilm formation under Mn 2+ deficiency and offered a new insight into enhancing LAB viability from biofilm perspective. This work provided strategies for industrial biofilm design and LAB efficient utilization. • MntR affects biofilm polysaccharide and protein, while SecA affects lipid and eDNA. • MntR regulates carbon/nitrogen balance to provide matrix and energy for biofilm. • SecA regulates amino acid metabolism/biosynthesis for biofilm structural maturation. • MntR and SecA affect purine and pyrimidine metabolism to regulate biofilm formation. • Fructose, thymine and xanthine may be the key metabolites in the biofilm formation.