Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.