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

Background Vitamin K ₂ (VK ₂ ), 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 ₂ 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 to produce menaquinol-8 (MKH ₂ -8, a reduced form of VK ₂ ) by overexpressing menA and ubiE , the rate-limiting enzymes of the menaquinol pathway. Secondly, we overexpressed different related genes wrbA , qorB and menF , respectively. Among these recombinant strains, the strain MUW reached a yield of 301.96 mg/L after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK ₂ . Subsequently, the rational metabolic engineering of gene knockout further increased the VK ₂ yield. The recombinant strain ΔB:MUW was selected as the dominant strain for further optimization, with a high VK ₂ yield of 723.59 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis. After 48 h cultivation, a high VK ₂ yield of 1355.29 mg/L was achieved by ΔB:MUWI in a 5 L fermenter. Conclusions This study demonstrates that metabolic engineering techniques combining rational modification of the metabolic pathway and optimization of gene expression can effectively cultivate strains with industrial competitiveness.