妥布霉素糖基转移酶基因tobM2的研究  

Research of glycosyltransferase gene tobM2 in tobramycin

作　　者：温淑平[1] 陈晖[1] 汪洋[1] 洪文荣[1] 

机构地区：[1]福州大学生物科学与工程学院,福建福州350116

出　　处：《延边大学学报（自然科学版）》2016年第1期39-44,共6页Journal of Yanbian University（Natural Science Edition）

基　　金：国家自然科学基金资助项目(31070093);国家"重大新药创制"科技重大专项项目(2012ZX09201101-008)

摘　　要：为探索妥布霉素生物合成过程中糖基转移的作用,对糖基转移酶基因tobM2进行了阻断研究.以妥布霉素生物合成基因簇为模板,构建了同源重组质粒pMB4,通过接合转移将质粒pMB4导入黑暗链霉菌Tt-49,然后利用红霉素抗性筛选并经PCR验证,获得了一株基因tobM2被阻断的工程菌TW402.发酵及代谢产物分析结果表明:工程菌总效价略有下降,约为出发菌株的85%.TLC检测与质谱分析显示,工程菌TW402主产安普霉素,并积累中间产物尼泊拉胺.TobM2转化尼泊拉胺生成妥布霉素,tobM2基因的缺失阻断了妥布霉素的生物合成,获得一株主产安普霉素工程菌.The glycosyltransferase gene tobM2 was disrupted to study the reaction of glycosylation in the tobramycin biosynthesis.A recombinant plasmid pMB4 was constructed with tobramycin biosynthesis gene cluster as the template.Then,the plasmid pMB4 was transformed into Streptomyces tenebrarius Tt-49 by conjugation.A mutant strain TW402 was obtained by the gene tobM2 was disrupted,screened and confirmed by erythromycin resistance and PCR amplification.Ferment and metabolite analysis showed that the total titer of mutant strain TW402 decreased slightly,which was about 85% of that of the wild-type strain.TLC detection and MS analyses indicated that the mutant strain TW402 mainly produced apramycin,and nebramine was accumulated.TobM2 converted nebramine into tobramycin,and apramycin-producing engineering bacterium was obtained by disruption of tobM2 gene,which blocked the tobramycin biosynthesis.

关 键 词：黑暗链霉菌 妥布霉素 tobM2 基因阻断 

分 类 号：Q93[生物学—微生物学]