摘要
目的阻断黑暗链霉菌中安普霉素和氨甲酰妥布霉素的合成,构建主产妥布霉素工程菌。方法以安普霉素生物合成基因apr J为靶标,克隆其上下游序列作为同源交换臂,构建重组质粒p XJ401,利用接合转移技术将其导入黑暗链霉菌Tt-49,获得工程菌TX302,并在此基础上敲除氨甲酰基转移酶基因tob Z,获得工程菌TX308。采用MS法,检测工程菌次级代谢产物的变化。结果 TX302主产氨甲酰妥布霉素,未检测到安普霉素。TX308不再合成氨甲酰妥布霉素,产物只检测到妥布霉素。结论磷酸变位酶基因apr J是安普霉素生物合成的关键基因,敲除后能有效阻断安普霉素的合成,工程菌TX308大量积累妥布霉素,不再合成安普霉素和氨甲酰妥布霉素,具有重要的产业化价值。
Objective To construct tobramycin overproducing strain by blocking the biosynthesis of carbamoyltobramycin and apramycin in Streptomyces tenebrarius. Methods The upstream and downstream of aprJ, a gene specific for the biosynthesis of apramycin, were cloned and used as exchange arms for homologous recombinant. The recombinant plasmid pXJ401 was conjugated into S. tenebrarius Tt49 to obtain engineering bacteria TX302. And then, tobZ coding carbamoyltransferase was disrupted to obtain engineering bacteria TX308. The change of secondary metabolites of TX302 and TX308 are detected by MS analysis. Results TX302 mainly produces carbamoyltobramycin, and no apramycin was detected in its secondary metabolites. However, TX308 produced tobramycin instead of carbamoyltobramycin. Conclusion block the biosynthesis of apramycin. TX308 overproduces production of tobramycin. The disruption of the phosphosugar mutase gene aprJ will tobramycin, and it can be used for industrial fermentative
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2014年第12期891-895,共5页
Chinese Journal of Antibiotics
基金
国家自然科学基金(No.31070093)
国家"重大新药创制"科技重大专项(No.2012ZX09201101-008)
关键词
黑暗链霉菌
安普霉素
妥布霉素
工程菌
Streptomyces tenebrarius
Apramycin
Tobramycin
Engineering bacteria
