Increasing Vitamin\nK<sub>2</sub> Synthesis in Bacillus subtilis by Controlling the Expression of\nMenD and Stabilizing MenA

As an indispensable\nmember of the family of lipid vitamins,\nvitamin\nK<sub>2</sub> (MK-7) plays an important role in blood coagulation,\ncardiovascular health, and kidney health. Microbial fermentation is\nfavored due to its high utilization rate of raw materials, simple\noperation, and moderate conditions. However, the biosynthesis pathway\nof vitamin K<sub>2</sub> in microorganisms is highly complex, which\nhinders its industrial production in microbial cell factories. One\nof the major challenges is the stable expression and deregulation\nof key enzymes in the vitamin K<sub>2</sub> biosynthesis pathway,\nwhich remains unclear and has undergone little investigation. In this\nstudy, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid\nsynthase (MenD) and 1,4-dihydroxy-2-naphthoate polyprenyltransferase\n(MenA) were identified as pivotal enzymes in the biosynthesis of vitamin\nK<sub>2</sub>. To investigate the catalytic efficiency of MenD in\nthe biosynthesis pathway of vitamin K<sub>2</sub>, structure-based\nmutation design and site-directed mutagenesis were performed. Three\nmutation sites were identified in MenD: A115Y, R96 M, and R323M, which\nimprove the expression level and protein stability. Meanwhile, the\nMenA mutant T290M, which exhibits improved protein stability, was\nobtained by modifying its hydrophobic stacking structure. Finally,\nan engineered strain noted ZQ13 that combinatorially overexpressed\nMenD (A115Y) and MenA (T290M) mutants was constructed and achieved\n338.37 mg/L vitamin K<sub>2</sub> production in a 3-L fermenter.