Combinatorial engineering for improved menaquinone-4 biosynthesis in Bacillus subtilis

Menaquinone-4 (MK-4), one form of vitamin K, plays an important role in cardiovascular and bone health. Menaquinone-4 (MK-4) is a valuable vitamin K2 that is difficult to synthesize organically, and now is mainly produced by microbial fermentation. Herein we significantly improved the synthesis efficiency of MK-4 by combinatorial pathway engineering in Bacillus subtilis 168, a model industrial strain widely used for production of nutraceuticals. The metabolic networks related to MK-4 synthesis include four modules, namely, MK-4 biosynthesis module, methylerythritol phosphate (MEP) module, mevalonate-dependent (MVA) isoprenoid module, and menaquinone module. Overexpression of menA, menG, and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module with strong constitutive promoter P-43 resulted in 8.1 +/- 0.2 mg/L of MK-4 (No MK-4 was detected in the wild-type B. subtilis 168). MK-4 titer was further increased by 3.8-fold to 31.53 +/- 0.95 mg/L by knockout of hepT gene, which catalyzes the conversion of Farnesyl diphosphate to Heptaprenyl diphosphate. In addition, simultaneous overexpression of dxs, dxr, and ispD-ispF genes in MEP module with strong promoter P-43 increased the titer of MK-4 to 78.1 +/- 1.6 mg/L. Moreover, expression of the heterogeneous MVA module genes (mvaKl, mvaK2, mvaD, mvaS, and mvaA) resulted in 90.1 +/- 1.7 mg/L of MK-4. Finally, in order to further convert the enhanced carbon metabolism flux to MK-4, simultaneous overexpression of the genes crtE, menA, and menG in menaquinone pathway with strong promoter P43 increased the titer of MK-4 to 120.1 +/- 0.6 mg/L in shake flask and 145 +/- 2.8 mg/L in a 3-L fed-batch bioreactor. Herein the engineered B. subtilis strain may be used for the industrial production of MK-4 in the future.