Menaquinone-7 (MK-7), an important bioactive form of vitamin K2, exhibits broad application prospects in promoting bone health, preventing cardiovascular diseases and treating neurodegenerative disorders.In this study, Bacillus subtilis 168 was selected as the chassis strain, and a multi-module metabolic engineering strategy was employed to optimize the MK-7 biosynthetic pathway systematically.Initially, key genes in competing branch metabolic pathways, including the isochorismate-pyruvate lyase gene (dhbB), aromatic amino acid biosynthesis genes (trpE/aroH), and the phosphotransferase system (PTS) glucose transporter gene (ptsG), were knocked out to minimize precursor diversion.Subsequently, the efficiency of the electron transport chain was enhanced by overexpressing the NADH dehydrogenase gene (ndh) and the terminal oxidase gene (sdhCAB), thereby facilitating electron transfer.Furthermore, the heterologous dxs gene from Agrobacterium tumefaciens was introduced, and the endogenous rate-limiting enzyme gene hepS was overexpressed to increase the flux of the methylerythritol 4-phosphate (MEP) pathway.Ultimately, the engineered high-yielding strain S13 achieved an MK-7 titer of (123.90±6.52) mg/L in shake-flask fermentation.Upon scale-up validation in a 3 L fermenter, the production further increased to 242.38 mg/L, laying a solid foundation for the industrial-scale microbial fermentation of MK-7.
GUO Fengming
,
ZHU Qiyao
,
DU Liping
,
FU Gang
,
ZHANG Dawei
. Metabolic engineering of Bacillus subtilis for efficient production of vitamin K2[J]. Food and Fermentation Industries, 2026
, 52(5)
: 1
-9
.
DOI: 10.13995/j.cnki.11-1802/ts.043152
[1] KUROSU M, BEGARI E.Vitamin K2 in electron transport system:Are enzymes involved in vitamin K2 biosynthesis promising drug targets?[J].Molecules, 2010, 15(3):1531-1553.
[2] SATO T, INABA N, YAMASHITA T.MK-7 and its effects on bone quality and strength[J].Nutrients, 2020, 12(4):965.
[3] YUAN Y, SZETO I M, LI N, et al.Effects of menaquinone-7 on the bone health of growing rats under calcium restriction:New insights from microbiome-metabolomics[J].Nutrients, 2023, 15(15):3398.
[4] TAN J, ZHU R, LI Y, et al.Vitamin K2 in managing nocturnal leg cramps:A randomized clinical trial[J].JAMA Internal Medicine, 2024, 184(12):1443-1447.
[5] EMEKLI-ALTURFAN E, ALTURFAN A A.The emerging relationship between vitamin K and neurodegenerative diseases:A review of current evidence[J].Molecular Biology Reports, 2023, 50(1):815-828.
[6] HARIRI E, KASSIS N, ISKANDAR J P, et al.Vitamin K2-a neglected player in cardiovascular health:A narrative review[J].Open Heart, 2021, 8(2):e001715.
[7] LAL N, BERENJIAN A.Cis and trans isomers of the vitamin menaquinone-7:Which one is biologically significant?[J].Applied Microbiology and Biotechnology, 2020, 104(7):2765-2776.
[8] FANG Z W, WANG L, ZHAO G H, et al.A simple and efficient preparative procedure for menaquinone-7 from Bacillus subtilis (natto) using two-stage extraction followed by microporous resins[J].Process Biochemistry, 2019, 83:183-188.
[9] 康倩, 向梦洁, 张大伟.枯草芽孢杆菌在系统与合成生物技术中研究进展及工业应用[J].生物工程学报, 2021, 37(3):923-938.
KANG Q, XIANG M J, ZHANG D W.Research progress and industrial application of Bacillus subtilis in systematic and synthetic biotechnology[J].Chinese Journal of Biotechnology, 2021, 37(3):923-938.
[10] YANG S M, CAO Y X, SUN L M, et al.Modular pathway engineering of Bacillus subtilis to promote de novo biosynthesis of menaquinone-7[J].ACS Synthetic Biology, 2019, 8(1):70-81.
[11] HUANG W, LI J L, ZHU Q Y, et al.Increasing vitamin K2 synthesis in Bacillus subtilis by controlling the expression of MenD and stabilizing MenA[J].Journal of Agricultural and Food Chemistry, 2024, 72(41):22672-22681.
[12] GAO X L, LUO Y N, ADINKRA E K, et al.Engineering a PhrC-RapC-SinR quorum sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis[J].Microbial Cell Factories, 2025, 24(1):88.
[13] SPIZIZEN J.Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[J].Proceedings of the National Academy of Sciences of the United States of America, 1958, 44(10):1072-1078.
[14] JOHNSTON J M, BULLOCH E M.Advances in menaquinone biosynthesis:Sublocalisation and allosteric regulation[J].Current Opinion in Structural Biology, 2020, 65:33-41.
[15] ROWLAND B M, TABER H W.Duplicate isochorismate synthase genes of Bacillus subtilis:Regulation and involvement in the biosyntheses of menaquinone and 2, 3-dihydroxybenzoate[J].Journal of Bacteriology, 1996, 178(3):854-861.
[16] GULDAN H, STERNER R, BABINGER P.Identification and characterization of a bacterial glycerol-1-phosphate dehydrogenase:Ni(2+)-dependent AraM from Bacillus subtilis[J].Biochemistry, 2008, 47(28):7376-7384.
[17] ZAGOREC M, POSTMA P W.Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis[J].Molecular and General Genetics MGG, 1992, 234(2):325-328.
[18] DING X M, ZHENG Z M, ZHAO G H, et al.Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis[J].Microbial Cell Factories, 2022, 21(1):101.
[19] DI X N, ORTEGA-ALARCON D, KAKUMANU R, et al.MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply[J].Plant Communications, 2023, 4(3):100512.
[20] GAO Q X, CHEN H, WANG G Y, et al.Highly efficient production of menaquinone-7 from glucose by metabolically engineered Escherichia coli[J].ACS Synthetic Biology, 2021, 10(4):756-765.
[21] SATO T, YAMADA Y, OHTANI Y, et al.Production of menaquinone (vitamin K2)-7 by Bacillus subtilis[J].Journal of Bioscience and Bioengineering, 2001, 91(1):16-20.
[22] BERENJIAN A, CHAN N L, MAHANAMA R, et al.Effect of biofilm formation by Bacillus subtilis natto on menaquinone-7 biosynthesis[J].Molecular Biotechnology, 2013, 54(2):371-378.
