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Background <p>Vitamin K<sub>2</sub> (VK<sub>2</sub>), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK<sub>2</sub> and the reduction of its production cost have become the focus of attention.</p> Results <p>In this work, an <i>E. coli</i> strain with high VK<sub>2</sub> accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant <i>E. coli</i> strain BW-T7/MU to produce menaquinol-8 (MKH<sub>2</sub>-8, a reduced form of VK<sub>2</sub>) by overexpressing <i>menA</i> and <i>ubiE</i> genes, which encoding the rate-limiting enzymes of the menaquinol pathway. After 24&#xa0;h and 48&#xa0;h of fermentation, this strain BW-T7/MU reach a titer of 303&#xa0;mg/L and 232&#xa0;mg/L. Secondly, we overexpressed different related genes <i>wrbA</i> (oxidative stress mitigation), <i>qorB</i> (reduction of quinones) and <i>menF</i> (conversion of chorismate to isochorismate), respectively. Among these recombinant strains, the strain BW-T7/MUW (overexpressing <i>menA</i>, <i>ubiE</i> and <i>wrbA</i> genes) reached the highest titer of VK<sub>2</sub> after 48&#xa0;h of fermentation. The optimization of the medium led to an increase in the accumulation of VK<sub>2</sub>. Subsequently, the rational metabolic engineering of gene knockout further increased the titer of VK<sub>2</sub>. The recombinant strain ΔB/MUW was selected as the dominant strain for further optimization, with a high VK<sub>2</sub> titer of 724&#xa0;mg/L. A final attempt is to overexpress <i>ispB</i> gene to increased flux of isoprenoid side chain synthesis, resulting in strain ΔB/MUWI with a titer of 859&#xa0;mg/L in a shake flask and 1360&#xa0;mg/L in a 5&#xa0;L fermenter after 48&#xa0;h cultivation.</p> Conclusions <p>The stepwise engineering strategy raised the VK<sub>2</sub> titer from the initial 303&#xa0;mg/L to 859&#xa0;mg/L through rational pathway modification and systematic gene expression. Further optimization in batch fermentation increased the VK<sub>2</sub> titer to 1360&#xa0;mg/L, which highlights the strong engineering impact of our strategy.</p>

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Metabolic engineering of Escherichia coli BW25113 for the production of Vitamin K2 based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月8日

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  • Changchuan Ye,
  • Yan Zhang,
  • Jie Zhang,
  • Menglei Shi,
  • Feixue Nie,
  • Qinghua Liu

摘要

Background

Vitamin K2 (VK2), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK2 and the reduction of its production cost have become the focus of attention.

Results

In this work, an E. coli strain with high VK2 accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant E. coli strain BW-T7/MU to produce menaquinol-8 (MKH2-8, a reduced form of VK2) by overexpressing menA and ubiE genes, which encoding the rate-limiting enzymes of the menaquinol pathway. After 24 h and 48 h of fermentation, this strain BW-T7/MU reach a titer of 303 mg/L and 232 mg/L. Secondly, we overexpressed different related genes wrbA (oxidative stress mitigation), qorB (reduction of quinones) and menF (conversion of chorismate to isochorismate), respectively. Among these recombinant strains, the strain BW-T7/MUW (overexpressing menA, ubiE and wrbA genes) reached the highest titer of VK2 after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK2. Subsequently, the rational metabolic engineering of gene knockout further increased the titer of VK2. The recombinant strain ΔB/MUW was selected as the dominant strain for further optimization, with a high VK2 titer of 724 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis, resulting in strain ΔB/MUWI with a titer of 859 mg/L in a shake flask and 1360 mg/L in a 5 L fermenter after 48 h cultivation.

Conclusions

The stepwise engineering strategy raised the VK2 titer from the initial 303 mg/L to 859 mg/L through rational pathway modification and systematic gene expression. Further optimization in batch fermentation increased the VK2 titer to 1360 mg/L, which highlights the strong engineering impact of our strategy.