Metabolic engineering of Escherichia coli BW25113 for the production of Vitamin K2 based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification
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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 VK 2 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 (MKH 2 -8, a reduced form of VK 2 ) 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 VK 2 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 titer of VK 2 . The recombinant strain ΔB/MUW was selected as the dominant strain for further optimization, with a high VK 2 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 VK 2 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 VK 2 titer to 1360 mg/L, which highlights the strong engineering impact of our strategy.
