Modular Pathway Engineering Of Bacillus Subtilis To Promote The Biosynthesis Of Menaquinone-7

Menaquinone-7(MK-7),one of vitamin K₂,plays an important role in the blood coagulation and the prevention of osteoporosis and cardiovascular calcification.Microbial production of MK-7 has the advantages of quality control,safety,greenness and naturalness,compared with the chemical synthesis of MK-7.The construction of genetic engineering strain has more advantages than mutation breeding.In this paper,Bacillus subtilis 168 was choosed as the starting strain,and the biosynthetic pathway of MK-7 was divided into four modules,namely,the MK-7 pathway,the shikimate(SA)pathway,the methylerythritol-4-phosphate(MEP)pathway,the glycerol dissimilation pathway.In situ replacement of the promoter of the men FDHBEC operon(MK-7 pathway)and overexpression of the heterologous gene menI had no significant effect on the synthesis of MK-7.However,overexpression of men A resulted in a 109%increase in MK-7 production to 19.21±0.19 mg/L.In situ replacement of the promoter of the hep S-men G-hep T operon could promote the synthesis of MK-7,nevertheless,in situ replacement of the promoter of this operon in strain overexpressing men A resulted in a slight decrease in MK-7 production.Overexpressions of the genes aroA,aroD,and aroE(SA pathway)resulted in a decrease in the yield of MK-7,while overexpression of aro K did not significantly promote the synthesis of MK-7.Overexpression of dxs,dxr,yacM,and yacN(MEP pathway)could promote the synthesis of MK-7,and simultaneous overexpression of these four genes increased MK-7 production by 83%.However,overexpression of ispE,yqfP,and yqiD(MEP pathway)resulted in a decrease in MK-7 production.Deletion of ldh and pab B-pab A had an unobvious effect on the synthesis of MK-7.Deletion of alsS-alsD and aroH resulted in a substantial decrease in the MK-7production.However,deletion of dhb B,cutting off the synthesis of enterobactin,increased the yield of MK-7 by 11%.In addition,overexpression of glpK and glpD(glycerol dissimilation pathway),and deletion of mgs A and ara M which blocked the synthesis of methylglyoxal and glycerol-1-phosphate,respectively,resulted in a 22%increase in MK-7 production.In addition,overexpression of the heterologous gene aroG^fbrand the endogenous gene pyrG^fbrresulted in a slight increase in MK-7 production.However,overexpression of HepS,the heptaprenyl diphosphate synthase component I,increased MK-7 production by 11.6%.Deletion of cydAB encoding the bd oxidase had no significant effect on MK-7 synthesis,while overexpression of Vitreoscilla hemoglobin VHb resulted in a slight increase in MK-7 production.Finally,the strain BSMK11was constructed,and its MK-7 yield of shake flask fermentation was 58.95±1.20mg/L,which was 5.4-fold higher than that of the starting strain.After 132 h of the fed-batch fermentation in a 5 L fermenter,the MK-7 titer was 281.4±5.0 mg/L(12.0mg/g DCW),which was higher than the highest yield of MK-7 produced by B.subilis natto.This study carried out a comprehensive engineering transformation for the de novo synthetic pathway of MK-7 in B.subtilis,with clear genetic background and ability to overcome the defects of mutant strains,which had important scientific significance and application value.