Enhancing MK-7 production in a Bacillus subtilis mutant strain via a two-stage leakage fermentation strategy and revealing metabolic mechanisms

Menaquinone-7 (MK-7), a subtype of Vitamin K 2 , is in high demand within the healthcare sector due to its health benefits. This study proposes a two-stage fermentation strategy combining submerged fermentation with leakage fermentation to enhance MK-7 production in a Bacillus subtilis mutant strain and preliminarily reveal its potential metabolic mechanisms. The Results showed that this strategy achieved a total MK-7 yield of 163.56 mg/L, representing an 85.48% increase over conventional biofilm fermentation, among which extracellular MK-7 production was significantly increased by 269.65%. Results from scanning electron microscopy, fluorescence microscopy, and flow cytometry confirmed that the enhanced MK-7 production was closely associated with increased membrane permeability induced by Brij-58. Furthermore, integrating gas chromatography–ion mobility spectrometry (GC-IMS) and untargeted metabolomics analyses revealed that the highly active central carbon metabolism in the first stage promoted cell growth and MK-7 precursor accumulation, while the possible diversion of carbon flux to secondary metabolic pathways in the second stage facilitated efficient MK-7 biosynthesis. Meanwhile, it was found that the increase in membrane permeability might be attributed to oxidative stress induced by Brij-58, thereby facilitating the efflux of intracellular metabolites and effectively alleviating excessive intracellular MK-7 accumulation. These findings could serve as a potential industrial reference for future process optimization and scale-up validation of MK-7 fermentation, while also offering useful insights for addressing yield bottlenecks in other microbial metabolites.