To improve acid tolerance in lactic acid bacteria, Leuconostoc mesenteroides subsp. mesenteroides KCTC 3718 producing only D-lactic acid was challenged with exogenously-supplied lactic acid. During the progress of the adaptive evolution to 100 g/L of lactic acid concentration, we had selected mutant strains exhibiting high half maximal inhibitory concentration (IC50) and lactic acid production in fermentation. Two mutant strains, LMS100A and LMS100B, isolated at 100 g/L lactic acid condition significantly increased by 2.1-fold the IC50 values compared to wild type. In fed-batch fermentation, LMS100A and LMS100B achieved 113.4 g/L and 99.6 g/L of the D-lactic acid productions, which is higher than 67.0 g/L lactic acid of wild type. In order to investigate mechanisms of the acid tolerance and changes between mutant strains, omics analysis were performed for wild type, LMS50, LMS60, LMS70A and LMS70B in which fitness probably exhibited a fast increase under acid stress. Analysis of the physiological data showed that the mutant cells retained higher intracellular ammonia concentrations and redox power, but not intracellular ATP levels. Moreover, the increased contents of F0F1 ATPase, phosphosglycerate mutase and several amino acids including alanine, glutamate, asparagine, valine and tyrosine may contribute to maintain intracellular pH homeostasis. In acid stress, results presented in the manuscript also suggested that F0F1 ATPase epsilon subunit deleted C-terminal structure may increase ATP hydrolysis activity to extrude proton from the cells and improve cell viability.
