Synergistic Optimization of Bacillus subtilis for Efficiently Producing Menaquinone-7 (MK-7) by Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Metabolic Engineering

Menaquinone-7 (MK-7) plays a crucial role in preventing fractures and certain cardiovascular diseases and is one of the essential vitamins in the human body. In this study, a strain of Bacillus subtilis that produces MK-7 was isolated from commercially available natto fermentation agents, with an MK-7 titer of 75 mg/L. It was named L-5. Firstly, by employing Atmospheric and Room Temperature Plasma (ARTP) mutagenesis technology and protoplast fusion techniques, mutants resistant to 1-hydroxy-2-naphthoic acid (HNA) and diphenylamine (DPA) were obtained, with the titer of MK-7 reaching 196 mg/L. It was named R-8. Based on whole-genome sequencing technology, four mutants involved in the MK-7 synthesis pathway of strain L-5 were identified: 2-succinyl-5-enol-pyruvate-6-hydroxy-3-cyclohexen-1-carboxylic acid, MenD (S249L); (1,4)-dihydroxy-2-naphthalic acid-heptaisoprenyltransferase, MenA (S196L); 1-deoxy-D-xylose-5-phosphate synthetase, Dxs (N60D, Q185H); and hydroxy acid reductive isomerase, Dxr (Q351K). The overexpression of these mutants led to increases in MK-7 production of 19 mg/L, 20 mg/L, 17 mg/L, and 16 mg/L, respectively, compared to the unmutated genes. These mutations have been shown to be effective. To further enhance the production of MK-7, the mutants menD (S249L), menA (S196L), Dxs (N60D, Q185H), and Dxr (Q351K) were co-expressed. The final titer of MK-7 reached 239 mg/L. This study provides theoretical support for the future genetic modification of key enzymes in the MK-7 biosynthetic pathway.