Vitamin K has emerged as a multi-functional vitamin that is essential in a diverse range of physiological effects in the human body. Among all the vitamin K family members, menaquinone-7 (vitamin K2) has gained the most popularity for the prevention and treatment of osteoporosis and atherosclerosis. The aim of this study was to develop a strategy to produce high level of MK-7 via fermentation by Bacillus subtilis for applications in the food and pharmaceutical industries.
A series of screening and optimisation steps were carried out to develop a viable MK-7 formula for fortified food application. The highest titre of 30 mg/L MK-7 was achieved by using 10 w/v % soy protein and 5 w/v% glycerol supplemented with 0.5 w/v % yeast extract and 0.6 % K2HPO4.The major problem of foam formation in stirred fermenter was tackled by using a novel strategy of controlled dual feeding of glycerol and soy protein simultaneously. The MK-7 concentration in the fermentation was increased by four fold, from 30 mg/L in a 100 mL shake flask to 112 mg/L in a 2 L fed-batch bioreactor.
Metabolic engineering was another approach undertaken to increase MK-7 production. 12 engineered B. subtilis 168 strains were constructed by individually overexpressing or co-overexpressing the rate limiting MEP pathway genes dxs, dxr, idi and the downstream gene menA in different orders. An ordered overexpression of menA-dxs-dxr-idi produced the highest titre of MK-7 at 50 mg/L in 50 mL SYG media in shake flasks. This improvement was 11-fold higher than the B. subtilis 168 control strain (4.5 mg/L) and 2.2-fold higher than the best-performing B. subtilis natto strain (22 mg/L).
In summary, the MK-7 production was significantly increased by process optimisation and metabolic engineering strategies in this study. This study also implies the potential of using B. subtilis as the cellular factory for production of other industrially important MEP pathway derived molecules.