Abstract: Vitamin K₂ (VK₂) plays a essential role in maintaining human bone and cardiovascular health. However, due to the silence or non-expression of some genes in the VK₂ synthesis pathway of lactic acid bacteria, VK₂ is negligible in dairy products. In this study, using Lactobacillus fermentum (LF1) as the starting strain, the lethality and the positive and negative mutation rates were analyzed under different ion beam injection conditions. The optimal parameters were determined to be an energy of 10 keV and a dose of 75(×2.6×10¹3 N⁺/cm²). Through successive ion beam iterations coupled with adaptive laboratory evolution (ALE), the high-yield VK₂ strain LFX was obtained, achieving a production rate of 0.67 mg/g. The strain’s genetic stability was confirmed through serial passage experiments. Transcriptome analysis revealed that in the VK₂-related synthesis pathway of LFX, genes associated with the biosynthesis of the naphthoquinone core and the polyprenyl side chain were markedly upregulated. Moreover, pathways involved in the antioxidant defense, cofactor biosynthesis, and central carbon and nitrogen metabolism were substantially enhanced. The findings suggested that iterative ion beam implantation and ALE could synergistically optimize metabolic fluxes, enhancing VK₂ biosynthesis and stability in the developed strains.

Key words: ion beam, mutagenesis breeding, adaptive laboratory evolution, vitamin K2;lactobacillus fermentum, transcriptome