Increasing Vitamin K₂ Synthesis in <i>Bacillus subtilis</i> by Controlling the Expression of MenD and Stabilizing MenA

As an indispensable member of the family of lipid vitamins, vitamin K₂ (MK-7) plays an important role in blood coagulation, cardiovascular health, and kidney health. Microbial fermentation is favored due to its high utilization rate of raw materials, simple operation, and moderate conditions. However, the biosynthesis pathway of vitamin K₂ in microorganisms is highly complex, which hinders its industrial production in microbial cell factories. One of the major challenges is the stable expression and deregulation of key enzymes in the vitamin K₂ biosynthesis pathway, which remains unclear and has undergone little investigation. In this study, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase (MenD) and 1,4-dihydroxy-2-naphthoate polyprenyltransferase (MenA) were identified as pivotal enzymes in the biosynthesis of vitamin K₂. To investigate the catalytic efficiency of MenD in the biosynthesis pathway of vitamin K₂, structure-based mutation design and site-directed mutagenesis were performed. Three mutation sites were identified in MenD: A115Y, R96 M, and R323M, which improve the expression level and protein stability. Meanwhile, the MenA mutant T290M, which exhibits improved protein stability, was obtained by modifying its hydrophobic stacking structure. Finally, an engineered strain noted ZQ13 that combinatorially overexpressed MenD (A115Y) and MenA (T290M) mutants was constructed and achieved 338.37 mg/L vitamin K₂ production in a 3-L fermenter.