Combinatorial metabolic engineering of Bacillus subtilis for menaquinone-7 biosynthesis.

Publisher: Wiley Country of Publication: United States NLM ID: 7502021 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1097-0290 (Electronic) Linking ISSN: 00063592 NLM ISO Abbreviation: Biotechnol Bioeng Subsets: MEDLINE

Publication: <2005->: Hoboken, NJ : Wiley
Original Publication: New York, Wiley.

Bacillus subtilis*/genetics ; Bacillus subtilis*/metabolism ; Metabolic Engineering*/methods ; Vitamin K 2*/metabolism ; Vitamin K 2*/analogs & derivatives; Metabolic Networks and Pathways/genetics

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction.
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22208122 National Natural Science Foundation of China; 32021005 National Natural Science Foundation of China

Keywords: Bacillus subtilis; combinatorial metabolic engineering; enzyme engineering; etiBsu1209; menaquinone‐7

11032-49-8 (Vitamin K 2)
8427BML8NY (menaquinone 7)

Date Created: 20240705 Date Completed: 20241009 Latest Revision: 20241009

20241009

10.1002/bit.28800

38965781