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庆大霉素C-6′位氨甲基化修饰基因的研究

胡育龙, 洪文荣

胡育龙, 洪文荣. 庆大霉素C-6′位氨甲基化修饰基因的研究[J]. 中国药科大学学报, 2015, 46(1): 105-110. DOI: 10.11665/j.issn.1000-5048.20150116
引用本文: 胡育龙, 洪文荣. 庆大霉素C-6′位氨甲基化修饰基因的研究[J]. 中国药科大学学报, 2015, 46(1): 105-110. DOI: 10.11665/j.issn.1000-5048.20150116
shu
HU Yulong, HONG Wenrong. C-6′aminomethylation modification in gentamicin biosynthesis gene cluster[J]. Journal of China Pharmaceutical University, 2015, 46(1): 105-110. DOI: 10.11665/j.issn.1000-5048.20150116
Citation: HU Yulong, HONG Wenrong. C-6′aminomethylation modification in gentamicin biosynthesis gene cluster[J]. Journal of China Pharmaceutical University, 2015, 46(1): 105-110. DOI: 10.11665/j.issn.1000-5048.20150116
shu

庆大霉素C-6′位氨甲基化修饰基因的研究

  • 福州大学生物科学与工程学院

基金项目: 国家自然科学基金资助项目(No.31070093)；国家“重大新药创制”科技重大专项资助项目(No.2012ZX09201101-008)

C-6′aminomethylation modification in gentamicin biosynthesis gene cluster

摘要

    摘要
  • 摘要: 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genT基因缺失的同源重组质粒pFT104，利用接合转移导入绛红色小单孢菌G1008，筛选获得一株genT基因缺失工程菌GT106(ΔgenT)。其次构建用于GT106上genN基因缺失的重组质粒pFTN203，通过接合转移导入GT106，筛选获得一株工程菌GTN205(ΔgenT+genN)。最后在genK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genK基因，筛选获得到一株工程菌GTNK308(ΔgenT+genN+genK)。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，genN基因缺失阻断了庆大霉素C1与C2b的合成，表明genN基因参与修饰绛红糖胺C-6′位氨甲基化，而genT并不参与修饰绛红糖胺C-6′位氨甲基化。
    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.
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    • 参考文献(14)
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  • 刊出日期:  2015-02-24

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