柔红霉素产生菌SIPI-DM中dnmV的基因置换

Gene Replacement of dnmV in Daunorubicin Producer SIPI-DM

摘要:

dnmV基因编码柔红霉素生物合成途径中TDP-柔红糖胺C4酮基还原酶。阻断基因组上的dnmV基因并导入源于阿维菌素生物合成途径的aveBIV基因可构建得到表柔红霉素工程菌。本文从高产的柔红霉素产生菌SIPI-DM中分别扩增dnmV基因两侧同源交换臂, 并在两侧交换臂中插入aveBIV基因构建用于置换dnmV基因的同源双交换重组质粒。经筛选及验证得到aveBIV基因直接置换dnmV基因的表柔红霉素工程菌, 且该工程菌基因组上不引入抗性基因, 有利于进一步的基因改造。

Abstract:

C4-keto reduction in the TDP-daunosamine pathway of daunorubicin biosynthesis was catalyzed by DnmV ketoreductase. Epidaunorubicin producer has been constructed previously by disruption of dnmV gene and integration of aveBIV originated from biosynthesis pathway of avermectin. In this work, dnrIJ and dnmZU were amplified from genome of SIPI-DM, a high-daunorubicin producer. Recombinant plasmid pYG838 was constructed with aveBIV insertion between these two genes, which used as homologous recombinant arms. After screening and validation, an epidaunorubicin producer with dnmV replacing by aveBIV which is genetically and metabolically stable was obtained. The lack of antibiotic resistant marker in chromosome of this mutant will be helpful for further gene disruption.