Metabolic engineering of Bacillus subtilis for high‐titer production of menaquinone‐7

Abstract Menaquinone‐7 (MK‐7) is a member of vitamin K 2 used for prevention from osteoporosis and cardiovascular calcification. This study constructed Bacillus subtilis strains for high‐titer production of MK‐7 through metabolic engineering approaches. In B. subtilis , MK‐7 biosynthesis was categorized into five modules: glycerol dissociation pathway, shikimate pathway, pyrimidine metabolic pathway, methylerythritol phosphate pathway, and MK‐7 pathway. Overexpression of GlpK and GlpD (glycerol dissociation pathway) led to a ~10% increase in the MK‐7 titer. Deletion of the genes mgsA and araM increased the MK‐7 production by 15%. Furthermore, overexpression of AroG D146N (shikimate pathway), PyrG E156K (pyrimidine metabolic pathway), HepS (methylerythritol phosphate pathway), and VHb could also increase the MK‐7 titer. Finally, we obtained a recombinant strain BSMK_11 with simultaneous overexpressing the genes glpK , glpD , aroG fbr , pyrG fbr , hepS , vgb , and knockouting the genes mgsA and araM , and the MK‐7 titer reached 281.4 ± 5.0 mg/L (i.e., 12.0 mg/g DCW) in a 5 L fermenter.