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Background <p>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, <i>Bacillus subtilis</i> BS016, has been engineered. However, its biosynthetic efficiency remains hindered by bottlenecks, such as low isoprenoid side-chain elongation efficiency.</p> Results <p>To address this limitation, a thermophilic farnesyl diphosphate (FPP) synthase from <i>Geobacillus stearothermophilus</i>, characterized by high activity and stability, was identified via database mining. This enzyme was modularly assembled with the endogenous <i>hepS</i>-<i>menG</i>-<i>hepT</i> operon in <i>B. subtilis</i> BS016, driven by the strong promoter P<sub>hbs</sub>. 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&#xa0;mg/L, representing an 11% increase, and maintained a stable titer of 87.9&#xa0;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&#xa0;mg/L.</p> Conclusion <p>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.</p>

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Directed screening and spatial coupling of farnesyl diphosphate synthase for enhancing menaquinone-7 production in Bacillus subtilis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月14日

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  • Xiumin Ding,
  • Rui Zhang,
  • Ying Liu,
  • Liang Hong,
  • Yalan Feng,
  • Qiang Li,
  • Zhiming Zheng,
  • Genhai Zhao

摘要

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 Phbs. 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.