Genetic Engineering And Process Optimization Of Bacillus Subtilis For Menaquinone-7 Production

Vitamin K₂?Menaquinone?is an essential vitamin existing in the daily diet,and Menaquinone-7?MK-7?is an important form of it.MK-7 can promote bone metabolism and the synthesis of blood coagulation factors.It can also prevent bone fractures and treat vitamin K deficiency hemorrhagic disease.At present,the chemical synthesis is the main method of industrially synthesizing MK-7.To harvest the biologically active MK-7,people need to further sort and purify the rough products,which increases the production cost.Microbial fermentation,however,can obtain the biologically active MK-7 directly,that simplifies the process and reduces the pollution.Bacillus subtilis,as a Gram-positive model microorganism,has a clear genetic background and mature genetic modification methods.In a recent work,we have engineered the synthesis modules of MK-7 in Bacillus subtilis 168 and the strain BS21 can produce 244 mg·L^-1 MK-7in shake flask.In BS21,the Methylerythritol-4-phosphate?MEP?pathway was supposed to be the limit part of MK-7 biosynthesis.The MEP pathway is the only way for Bacillus subtilis to synthesize the side chain structure of MK-7,so the expression intensity of the MEP pathway has an important influence on the biosynthesis of MK-7.In this study,BS21 was used as the starting strain to study the best expression intensity of genes of the MEP pathway.The results are as follows,?1?Using the constitutive promoter P₄₃ with higher expression intensity,we combined the five genes in the MEP pathway with different expression intensities.Twenty-five recombinant strains with different expression strength of MEP pathway genes were obtained.The recombinant strain BS21DFH produced 370 mg·L^-1 of MK-7 in the 7-day shake flask fermentation experiment.Subsequently,we enhanced the expression intensity of the key genes of the MEP pathway as ispE,ispG and ispH by using the constitutive promoter P₅₅₆ with high expression intensity,resulting in 4 recombinant strains.The MK-7 titer of BS21DFH5G in the7-day shake flask fermentation experiment reached 394 mg·L^-1,which was 61%higher than the starting strain BS21.?2?Optimizing the shake flask fermentation medium of MK-7 high-yield strain BS21DFH5G and the medium formula was set as glucose 30 g·L^-1,sucrose 70 g·L^-1,soybean peptone 50 g·L^-1,KH₂PO₄ 0.06 g·L^-1.Under this condition,the MK-7 titer of the recombinant strain BS21DFH5G was 403 mg·L^-1.Next,we optimized the fermentation process parameters?initial pH,temperature and aeration?in an 8-unit parallel bioreactor system consisting of 300mL glass vessels.The highest MK-7 titer of BS21DFH5G in the 300 mL reactor was 188mg·L^-1.?3?Based on this,we scaled-up the MK-7 production by the strain BS20DFH5G in the5 L and 15 L bioreactor,repectively.The MK-7 titer reached 199 mg·L^-1 in the 5 L bioreactor while the highest MK-7 titer reached 245 mg·L^-1 in the 15 L bioreactor after 120 hours.