庆大霉素生物合成基因簇中抗性基因gmrB及gmrA的功能研究
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摘要
目的研究绛红色小单孢菌(Micromonospora purpurea)G1008中抗性基因gmrB与gmrA功能,为阐明庆大霉素生物合成机制奠定基础。方法以红霉素抗性基因ermE分别置换gmrB和gmrA基因,构建突变株GerB102和GerA102。借助TLC和MS分析突变株代谢产物是否变化,同时检测突变株耐受自身代谢产物的能力是否发生变化。结果突变株GerB102代谢产物没有发生明显变化,仍主要积累庆大霉素C化合物,而GerA102代谢产物发生变化。庆大霉素耐受性试验结果显示,GerB102和G1008耐庆大霉素浓度达到6000U/m L以上,而GerA102不足50U/m L。结论 gmrB既非庆大霉素生物合成的关键基因,也非关键的抗性基因,而gmrA是庆大霉素抗性的关键基因。
Objective Characterizing the functions of gmrB and gmrA of Micromonospora purpurea G1008 to elucidate the gentamicin biosynthetic mechanisms. Methods The mutant strains GerB102 and GerA102 were constructed by replacing of gmrB and gmrA with the erythromycin resistance gene erm E, respectively. Then the possible alterations of the metabolites in these mutants were analyzed by TLC and MS. The resistance of the mutants was also detected against their own metabolites(gentamicin), respectively. Results The metabolite profile of GerB102 was not significantly changed where gentamicin C components still accumulated, while the metabolite profile of GerA102 was changed. Similar to the native G1008 strain, the tolerance to gentamicin of GerB102 reached 6,000 U/m L as well. Comparably, the tolerance to gentamicin of GerA102 was less than 50 U/m L. Conclusion The gmrB is neither a key gene for gentamicin biosynthesis nor an essential gene for gentamicin resistance, but the gmrA is a key gene responsible for the resistance to gentamicin.
Objective Characterizing the functions of gmrB and gmrA of Micromonospora purpurea G1008 to elucidate the gentamicin biosynthetic mechanisms. Methods The mutant strains GerB102 and GerA102 were constructed by replacing of gmrB and gmrA with the erythromycin resistance gene erm E, respectively. Then the possible alterations of the metabolites in these mutants were analyzed by TLC and MS. The resistance of the mutants was also detected against their own metabolites(gentamicin), respectively. Results The metabolite profile of GerB102 was not significantly changed where gentamicin C components still accumulated, while the metabolite profile of GerA102 was changed. Similar to the native G1008 strain, the tolerance to gentamicin of GerB102 reached 6,000 U/m L as well. Comparably, the tolerance to gentamicin of GerA102 was less than 50 U/m L. Conclusion The gmrB is neither a key gene for gentamicin biosynthesis nor an essential gene for gentamicin resistance, but the gmrA is a key gene responsible for the resistance to gentamicin.
引文
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[8]简心韵,邓子新,孙宇辉.氨基糖苷抗生素庆大霉素:基础研究的新进展及应用研究的新潜力[J].生物工程学报,2015,31(6):829-844.
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[12]严凌斌,洪文荣,方志锴.绛红色小单孢菌G1008接合转移体系的构建[J].中国抗生素杂志,2011,36(12):899-904.
[13]张国华,洪文荣,沈剑锋,等.依纽小单孢菌接合转移体系的构建[J].生物技术通报,2011,11:216-220.
[14]万云凤,洪文荣,石贤爱.西索米星生物合成基因sis I功能的研究[J].延边大学学报(自然科学版),2016,42(2):130-135.
[15]国家药典委员会.中华人民共和国药典[S].(2010年版第二部).北京:中国医药科技出版社,2010:附录93-98.
[2]Krasnova T P,Laznikova,T N,Likina,E V,et al.Role of cobalt in the biosynthesis of the components of the gentamicin complex[J].Antibiotiki,1978,23(1):12-18.
[3]王梓,孔令聪,贾博岩.氨基糖苷类抗生素耐药机制研究进展[J].中国兽药杂志,2015,49(3):65-69.
[4]Hong W R,Yan L B.Identification of gnt K,a gene required for the methylation of purpurosamine C-6'in gentamicin biosynthesis[J].J Gen Microbiol,2012,58(2):349-356.
[5]Kim H J,Mc Carty R M,Ogasawara Y,et al.Gen K-catalyzed C-6'methylation in the biosynthesis of gentamicin:Isolation and characterization of a cobalamin-dependent radical SAM enzyme[J].J Am Chen Soc,2013,135(22):8093-8096.
[6]蒋明星,鲁涛,文孟良.庆大霉素的生物合成[J].中国抗生素杂志,2016,41(1):16-25.
[7]Ni X P,Zong T T,Zhang H Y,et al.Biosynthesis of3-demethyl-gentamicin C components by gen N disruption strain of Micromonospora echinospora and test their antimicrobial activities in vitro[J].Microbiol Res,2016,185:36-44.
[8]简心韵,邓子新,孙宇辉.氨基糖苷抗生素庆大霉素:基础研究的新进展及应用研究的新潜力[J].生物工程学报,2015,31(6):829-844.
[9]Unwin J,Standage S,Alexander D,et al.Gene cluster in Micromonospora echinospora ATCC15835 for the biosynthesis of the gentamicin C complex[J].J Antibiot,2004,57(7):436-445.
[10]Moric I,Bajkic S,Savic M,et al.Heterologous Escherichia coli expression,purification and characterization of the Grm A aminoglycoside-resistance methyltransferase[J].Protein J,2009,28:326-332.
[11]金梦.庆大霉素生物合成中抗性基因gmr A及抗性相关基因的研究[D].武汉:华中农业大学,2015.
[12]严凌斌,洪文荣,方志锴.绛红色小单孢菌G1008接合转移体系的构建[J].中国抗生素杂志,2011,36(12):899-904.
[13]张国华,洪文荣,沈剑锋,等.依纽小单孢菌接合转移体系的构建[J].生物技术通报,2011,11:216-220.
[14]万云凤,洪文荣,石贤爱.西索米星生物合成基因sis I功能的研究[J].延边大学学报(自然科学版),2016,42(2):130-135.
[15]国家药典委员会.中华人民共和国药典[S].(2010年版第二部).北京:中国医药科技出版社,2010:附录93-98.