庆大霉素C-6'位氨甲基化修饰基因的研究

C-6' aminomethylation modification in gentamicin biosynthesis gene cluster

探索庆大霉素C-6'位氨甲基化修饰作用的基因。首先构建用于gen T基因缺失的同源重组质粒p FT104,利用接合转移导入绛红色小单孢菌G1008,筛选获得一株gen T基因缺失工程菌GT106(Δgen T)。其次构建用于GT106上gen N基因缺失的重组质粒p FTN203,通过接合转移导入GT106,筛选获得一株工程菌GTN205(Δgen T+gen N)。最后在gen K基因已经明确为C-6'位甲基化酶基因的基础上,在GTN205中敲除gen K基因,筛选获得到一株工程菌GTNK308(Δgen T+gen N+gen K)。工程菌发酵产物经质谱分析结果表明,与出发菌G1008相比,GT106组分未发生变化,而GTN205不再合成庆大霉素C1,产物主要积累在庆大霉素C1a和C2,GTNK308未检测到庆大霉素C2b。结果说明,gen N基因缺失阻断了庆大霉素C1与C2b的合成,表明gen N基因参与修饰绛红糖胺C-6'位氨甲基化,而gen T并不参与修饰绛红糖胺C-6'位氨甲基化。

This study explored the C-6＇ aminomethylation modification in gentamicin biosynthesis gene cluster. Plasmid p FT104 used for the gen T in-frame deletion was constructed and transformed into M. purpurea G1008 by conjugation. Mutant M. purpurea GT106（ Δgen T） confirmed by apramycin resistance and PCR amplification was then obtained. Next,a recombinant plasmid p FTN203 was constructed for blocking-up gen N. p FTN203 was introduced into M. purpurea GT106 by conjugation. A desired mutant strain GTN205（ Δgen T ＋ gen N） was obtained by PCR analysis. Finally,based on the role of gen K in C＇-6 methylation,the mutant strain GTNK308（ Δgen T ＋ gen N ＋gen K） was selected by knocking out gen K. Metabolites were isolated from the fermentation broths of mutant strains and analyzed by MS. The results indicated that the metabolites of M. purpurea GT106（ Δgen T） did not change compared with the original strain G1008. The mutant strain GTN205（ Δgen T ＋ gen N） no longer produced gentamicin C1,and yet accumulated in gentamicin C1 a and C2. Gentamicin C2 b was no longer detected in the metabolites of strain GTNK308（ Δgen T ＋ gen N ＋ gen K）. These results showed that the inactivation of gen N led to the blocking of the synthesis of gentamicin C1 and C2 b. It suggested that gen N might participate in the modification of aminomethylation at C-6＇of purpurosamine,while gen T was not involved in the C-6＇aminomethylation.