Abstract: In order to increase the yield of vitamin K₂(VK₂) during the fermentation of Bacillus natto,Bacillus natto SD-3 was used as the starting strain to construct an auxotrophic strain with high yield of VK₂ through chemical and physical mutagenesis. First,Bacillus natto SD-3 was subjected to diethyl sulfate(DES) mutagenesis and ultraviolet mutagenesis. the mutagenic conditions of SD-3 lethality mutagenesis standard was obtained by single factor test. Then the content of VK₂ in the fermentation broth were continuously screened and detected for several times. Finally the fermentation conditions were optimized by response surface analysis. The results showed that a double-auxotrophic VK₂ high-yield strain PT-6 with a branching acid-Trp/Phe/Tyr metabolic pathway and Phe^- and Trp^- synthetic defects was obtained,and the average yield of VK₂ reached 40.23 mg/L. The mutagenic conditions were:UV irradiation distance 25 cm,irradiation time 50 s,2% DES,and the reaction 60 min. The optimal fermentation conditions were:the volume of liquid 64 mL,the inoculation amount 3%,the temperature 38℃,and the VK₂ content reached 51.23 mg/L. Compared with the original strain SD-3,the final yield of VK₂ improved 134.35%.