Advances in the synthesis of menaquinone using microbial cell factories

Menaquinone, also known as vitamin K-2, is an essential vitamin. It contains a series of compounds; it has a 2-methyl-1,4-naphthoquinone ring as the parent ring, and different numbers of isoprene unit side chains at its C3 position. The common forms of K-2 are menaquinone-4, menaquinone-7 and menaquinone-8. Menaquinone can effectively prevent and treat vitamin K-2 deficiency-associated bleeding disorders, osteoporosis, cardiovascular disease and chronic kidney disease. Therefore, it has important applications in the fields of food and medicine. And the market demand for menaquinone is growing year by year with increased research on its function, and it is expected to reach a hundred-billion-dollar market. There are two production methods of menaquinone: Chemical synthesis and biosynthesis. The large-scale application of chemical synthesis is limited for the disadvantages of low yield and complicated separation process. And biosynthesis uses microorganisms that can naturally synthesize menaquinone to produce menaquinone, whichhas the advantages of simple operation and easy product separation. Therefore, biosynthesis has a better application prospect. In this review, we first summarise the biosynthetic process and metabolic pathways of menaquinone, including the research progress of the substrate uptake, shikimate, terpenoid backbone and menaquinone pathways. Then, metabolic engineering strategies, which have been applied in cell factories, such as Escherichia coli and Bacillus subtilis, are summarised, including overexpression, heterologous MVA synergy, knockdown and dynamic regulation. Additionally, we have introduced the yield of menaquinone from constructed microbial cell factories. Finally, we discuss the value of the two types of cell factories for industrial applications. E. coli has achieved a yield of 1.35 g/L menaquinone-7, the highest yield known for constructed cell factories. However, the application permission of menaquinone passed in China requires that its source is from B. subtilis, and the maximum yield of menaquinone-7 is only 310 mg/L in B. subtilis, which is of limited value for industrial application. Therefore, there is a need to further improve the menaquinone synthesis capacity of B. subtilis. Based on the results of applying metabolic engineering strategies, future research on metabolic engineering of cell factories should focus on the following points. Firstly, to find new metabolic bottlenecks in modified cell factories and enhance the metabolic flux of rate-limiting reactions.Classic overexpression solves the problem of insufficient metabolic flux in the rate-limiting step, but also causes other reactions to become new bottlenecks that limit product synthesis. Secondly, to expand the product storage space of menaquinone, the cell membrane of B. subtilis could be modified, or the intracellular lipid-soluble space could be expanded. Fat-soluble menaquinone-7 can only exist in the fat-soluble space of cell factories, and membrane modification in E. coli has proved that expanding the fat-soluble space can promote the synthesis of menaquinone-7. Thirdly, to find new targets to promote menaquinone synthesis at the global regulatory level to maximise the production performance of cell factories. The biosynthesis of menaquinone is complex, involving metabolic engineering of many pathways. And these metabolic engineering strategies often cause metabolic imbalance of cell factories. Therefore, it is a good way to look for regulators that promote the synthesis of menaquinone-from the global metabolic level of cell factories.