Abstract
BACKGROUND: Menaquinone-7 (MK-7), a highly bioactive form of vitamin K₂ with a long half-life, plays pivotal roles in preventing osteoporosis and cardiovascular diseases. A metabolically balanced MK-7-producing strain, Bacillus subtilis BS016, has been engineered. However, its biosynthetic efficiency remains hindered by bottlenecks, such as low isoprenoid side-chain elongation efficiency. RESULTS: To address this limitation, a thermophilic farnesyl diphosphate (FPP) synthase from Geobacillus stearothermophilus, characterized by high activity and stability, was identified via database mining. This enzyme was modularly assembled with the endogenous hepS-menG-hepT operon in B. subtilis BS016, driven by the strong promoter P(hbs). A synergistic expression cassette was constructed to achieve spatial co-localization of FPP synthesis with downstream isoprenoid side-chain elongation. The resulting engineered strain BS018 achieved a flask yield of 91.1 mg/L, representing an 11% increase, and maintained a stable titer of 87.9 mg/L in a 5-L fermenter. Further optimisation of fermentation conditions (liquid loading, 50 mL/500 mL; initial pH, 7.0; inoculum dose, 8%) ultimately elevated a flask yield to 109.6 mg/L. CONCLUSION: This study demonstrates that the directed screening of high activity heterologous enzymes, combined with the spatial co-assembly of endogenous pathways, can effectively reconstruct and enhance metabolic flux. This integrated strategy, combining directed enzyme screening with spatial pathway assembly, provides a generalizable framework for constructing efficient cell factories not only for vitamin K₂ but also for other high-value terpenoids. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-026-02930-1.