Modular Pathway Engineering of Bacillus subtilis To Promote De Novo Biosynthesis of Menaquinone-7

Menaquinone-7 (MK-7), a valuable vitamin K-2, plays an important role in the prevention of osteoporosis and cardiovascular calcification. We chose B. subtilis 168 as the chassis for the modular metabolic engineering design to promote the biosynthesis of MK-7. The biosynthetic pathway of MK-7 was categorized into four modules, namely, the MK-7 pathway (Module I), the shikimate (SA) pathway (Module II), the methylerythritol phosphate (MEP) pathway (Module III), and the glycerol metabolism pathway (Module IV). Overexpression of menA (Module I) resulted in 6.6 +/- 0.1 mg/L of MK-7 after 120 h fermentation, which was 2.1-fold that of the starting strain BS168NU (3.1 +/- 0.2 mg/L). Overexpression of aroA, aroD, and aroE (Module II) had a negative effect on the synthesis of MK-7. Simultaneous overexpression of dxs, dxr, yacM, and yacN (Module III) enabled the yield of MK-7 to 12.0 +/- 0.1 mg/L. Moreover, overexpression of glpD (Module IV) resulted in an increase of the yield of MK-7 to 13.7 +/- 0.2 mg/L. Furthermore, deletion of dhbB reduced the consumption of the intermediate metabolite isochorismate, thus promoting the yield of MK-7 to 15.4 +/- 0.6 mg/L. Taken together, the final resulting strain MK3-MEP123-Gly2-Delta dhbB with simultaneous overexpression of menA, dxs, dxr, yacM-yacN, glpD and deletion of dhbB enabled the yield of MK-7 to 69.5 +/- 2.8 mg/L upon 144 h fermentation in a 2 L baffled flask.