Abstract
BACKGROUND: Product inhibition is one of the major problems in lactic acid (LA) fermentation. Our previous study revealed that Bacillus coagulans 2-6 was an efficient producer of high-optical-purity L-LA. Its mutant strain B. coagulans Na-2 has better resistance to sodium lactate stress but the resistance mechanism has not been understood. RESULTS: In this study, the whole-genome sequencing of B. coagulans Na-2 was performed and one mutant gene mfs coding for the major facilitator superfamily (MFS) protein was revealed by comparative genome analysis. Ten mutation sites were identified between the wild (MFS-2-6) and mutant (MFS-Na-2) proteins, among which T127A and N154T were predicted locating in the center of the transmembrane transport channel. The MFS-2-6 and MFS-Na-2 were expressed separately in a genetically operable strain, B. coagulans DSM1, using the genes' native promoter. The expression of the two MFS proteins had no effect and a negative effect on L-LA production when the pH was controlled at 6.0 and 7.0 by sodium hydroxide, respectively. However, 4.2 and 4.6-fold of L-LA concentrations were obtained at pH 5.0 by the strains expressing MFS-2-6 and MFS-Na-2 than that by the control strain, respectively. The intracellular pH values of the strains expressing MFS-2-6 and MFS-Na-2 were approximately 0.69 and 0.45 higher than that of the control strain during pH-controlled fermentation at 5.0. Results suggest that the expression of MFS-2-6 and MFS-Na-2 were both conducive to L-LA production at low pH, while the better performance of the latter was probably due to the more appropriate intracellular pH during the whole fermentation process. CONCLUSIONS: The MFS protein identified here can improve the ability of B. coagulans to resist acidic environments and produce more L-LA at low pH. The MFS protein has an application potential in environment-friendly L-LA production.