Introduction

Menaquinone-7 (Figure 1) has a wide range of biological functions in the human body, such as treating Parkinson’s disease and osteoporosis. Therefore, menaquinone-7 has strong application prospects and possesses high market value in food, health,and medicine. Since the content of menaquinone-7 in natural food is extremely low, menaquinone-7 is mainly produced by fermentation of Bacillus species. B. subtilis is considered the dominant and most promising potential strain for microbial production of menaquinone-7 due to its long history of research and high content of menaquinone-7. However, the process of fermentation by B.subtilis usually takes a long time (approximately six days). Although metabolic engineering of B. subtilis for menaquinone-7 continues to progress, the productivity of MK-7 is low, and the fermentation cost of B. subtilis remains high, which keeps the global price of MK-7 high.[1]

Menaquinone-7 production in engineered Escherichia coli

Escherichia coli, which is fast-growing and easy to manipulate, has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Engineering E. colito synthesize menaquinone-7 is attractive and may provide an alternative method for economical industrial production of menaquinone-7.Compared with B. subtilis, E. coli mainly synthesizes ubiquinone-8 (Q-8) under aerobic conditions and mainly producesmenaquinone-8 (MK-8) instead of MK-7 under anaerobic conditions, since it contains octaprenyl diphosphate (OctPP) synthase (IspB) and does not contain HepPPS. Previous studieshave shown that E. coli can synthesize 290 μg MK-8/g WCWunder anaerobic conditions through metabolic engineering(Kong and Lee 2011). Then, we speculated that menaquinone-7 mightbe robustly produced in E. coli by overexpression of heterogenous HepPPS. However, anaerobic fermentation of E. coli ismore complicated and costly than aerobic fermentation. Therefore, the metabolic engineering of E. coli to synthesize menaquinone-7 under aerobic conditions is more practical.

In this study, to develop engineered Escherichia coli strains for menaquinone-7 production, heterogeneous heptaprenyl pyrophosphate synthetase (HepPPS) was introduced, and menaquinone-7 production was first achieved in engineered E. coli by over expression of Bacillus subtilis-derived HepPPS (BsHepPPS). Then, by optimizing the enzyme expression of the heterogenous mevalonic acid(MVA) pathway and the BsHepPPS, the titre of MK-7 increased to 2.3 μM, which was 22-fold higher than that of the original strain. The competitive pathways of MK-7 were further investigated by deletion of ubiCA or ispB. Finally, the scale-up fermentation of the engineered E. coli in a 5-L fermenter was studied under aerobic conditions using glucose, and 13.6 μM(8.8 mg/L) MK-7 was achieved. Additionally, metabolite analysis revealed a new bottleneck in the MK-7 pathway at ubiE,suggesting an avenue for further optimization.[1]

Advantages of menaquinone-7

Osteocalcin has been used as a biomarker for bone metabolism. Vitamin K deficiency leads to an increase in serum ucOC, and a high serum ucOC level has been associated with hip fractures,and has been recognized as an independent risk factor for fractures. Since 2007, serum ucOC has been used as a diagnostic marker to evaluate vitamin K deficiency in bones in Japan. A smaller dose of menaquinone-7 can γ-carboxylate OC compared to doses of K1 or MK-4. A supplemental intake of 250–1000μg/day of vitamin K1 activates OC, which is higher than the current RDIs of vitamin K in most countries. A markedly higher dose of MK-4 (600–1500 μg/day) is required to activate OC , as it has been shown to have a very short half-life in humans and it is poorly absorbed. Nutritional doses of MK-4, such as the consecutive intake of 60 μg/day or a single intake of 420 μg, have shown tobe ineffective. In contrast, menaquinone-7 at doses around the current RDI (90–180 μg/day) promoted OC carboxylation. A study demonstrated that menaquinone-7 derived from nat to has a very long half-life in the serum and induces more complete carboxylation of OC compared to vitamin K1 in humans. As all vitamin K homologs are converted to MK-4 in tissues, MK-4 is considered to perform other specific functions other than γ-carboxylation of VKDPs. However, in our previous study,the intake of a nutritional dose of MK-4 did not lead to an increase in MK-4 levels in the extra hepatic organs of rats, while that of menaquinone-7 led to a significant increase in MK-4 in organs such as the femur,brain, and testis. This implies that to achieve MK-4-specific physiological effects, it might be better to intake MK-7 as a MK-4 precursor than MK-4 itself.In addition to γ-carboxylation of VKDPs and the SXR-receptor ligand by conversion of menaquinone-7 to MK-4, menaquinone-7, the precursor of MK-4, directly activates bone formation by osteoblasts and suppresses bone resorption. It has also been shown that menaquinone-7 stimulates osteoblastogenesis and suppresses osteoclastogenesis by inhibiting the activation of NF-κB.[2]

References

[1] Gao Q, Chen H, Wang W, Huang J, Tao Y, Lin B. Menaquinone-7 production in engineered Escherichia coli. World J Microbiol Biotechnol. 2020;36(9):132. Published 2020 Aug 1. doi:10.1007/s11274-020-02880-9

[2] Sato T, Inaba N, Yamashita T. MK-7 and Its Effects on Bone Quality and Strength. Nutrients. 2020;12(4):965. Published 2020 Mar 31. doi:10.3390/nu12040965