Abstract: This study explored the C-6′aminomethylation modification in gentamicin biosynthesis gene cluster. Plasmid pFT104 used for the genT in-frame deletion was constructed and transformed into M. purpurea G1008 by conjugation. Mutant M. purpurea GT106(ΔgenT)confirmed by apramycin resistance and PCR amplification was then obtained. Next, a recombinant plasmid pFTN203 was constructed for blocking-up genN. pFTN203 was introduced into M. purpurea GT106 by conjugation. A desired mutant strain GTN205(ΔgenT+genN)was obtained by PCR analysis. Finally, based on the role of genK in C′-6 methylation, the mutant strain GTNK308(ΔgenT+genN+genK)was selected by knocking out genK. Metabolites were isolated from the fermentation broths of mutant strains and analyzed by MS. The results indicated that the metabolites of M. purpurea GT106(ΔgenT)did not change compared with the original strain G1008. The mutant strain GTN205(ΔgenT+ genN)no longer produced gentamicin C1, and yet accumulated in gentamicin C1a and C2. Gentamicin C2b was no longer detected in the metabolites of strain GTNK308(ΔgenT+genN+genK). These results showed that the inactivation of genN led to the blocking of the synthesis of gentamicin C1 and C2b. It suggested that genN might participate in the modification of aminomethylation at C-6′of purpurosamine, while genT was not involved in the C-6′aminomethylation.