A "high-efficient, low-carbon, cofactor recycling" menaquinone-7 (MK-7) synthetic strain was constructed by researchers from the Hefei Institutes of Physical Science (HFIPS) of the Chinese Academy of Sciences, laying a foundation for large-scale production of high-value MK-7 and terpenoids, according to study published in Microbial Cell Factories.

Mk-7 is a lipid-soluble vitamin K2 that plays an important role in the clotting cascade, maintenance of bone metabolism, prevention of arteriosclerosis, regulation of inflammation and neuroprotection.

In this study, the research team tried a different strategy to improve the production of MK-7. This is a bottom-up approach to synthetic biology.

After three rounds of Design-Build-Test-Learn cycle, the researchers reconstructed the pathways of MK-7 synthesis in Bacillus subtilis, promoted the central carbon metabolism flow to the MK-7 synthesis module, and balanced intracellular reducing force metabolism.

The classical gene editing technology Cre/Loxp was mainly used to replace the native promoters of key enzymes and rate-limiting enzymes in the MK-7 synthesis pathway with constitutive strong promoters, and knock out the competitive bypass metabolism, realizing "open source and throttling".

Moreover, they calculated the transformation of matter and energy in the pathway of synthesizing MK-7 from glycerol in Bacillus subtilis, and found that there is a cofactor imbalance in the synthesis of MK-7. After rational design of cofactor regeneration pathway, they introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis.

By testing the fermentation performance of the constructed MK-7 recombinant bacteria, it was found that it had excellent MK-7 synthesis ability and reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168.

Additionally, the balance of cofactors not only improves the synthesis efficiency of MK-7 in Bacillus subtilis, but also reduces the synthesis of by-products such as lactic acid, which decreases energy loss and improves carbon conversion.

This work was supported by the National Key R&D Program, the Anhui Provincial Science and Technology Major Project, and the Anhui Provincial Key R&D Program.

Pathways of MK-7 synthesis in B. subtilis 168. (Image by DING Xiumin)