在东方拟无枝酸菌中建立I-SceI内切酶介导的无标记敲除系统  

Construction meganuclease I-Sce I assisted the markerless deletion system in Amycolatopsis orientalis

作　　者：朱丽[1,2] 饶敏[2] 罗敏玉[2] 

机构地区：[1]上海交通大学药学院,上海200240 [2]上海来益生物药物研究开发中心有限责任公司,上海200240

出　　处：《中国抗生素杂志》2015年第5期330-333,共4页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(No.81202440)

摘　　要：目的在东方拟无枝酸菌中建立由I-Sce I介导的无标记高效重组系统。方法首先构建可用于东方拟无枝酸菌的I-Sce I操作盒,通过敲除多烯类化合物ECO-0501合成相关基因hem A2,比较操作盒使用前后获得hem A2基因双交换突变株的效率。结果在没有引入I-Sce I的情况下,随机重组的发生几率仅约2%,而导入后,由于I-Sce I酶对引入DNA位点的双链切割,导致双交换重组的发生率提高至90%以上。结论 I-Sce I系统是一种可用于东方拟无枝酸菌无标记遗传操作的有效工具。Objective To introduce the I-SceI restriction modification system into the Amycolatopsis orientalis for high efficiency recombination by DNA double strand break with I-SceI. Methods Firstly, the shuttle vector pLYZL102 with 18bp I-SceI recognized sites and the plasmid pLYZL104 with sceS gene following ermP promoter were constructed, and the up and down stream fragments of hemA2 gene for ECO-0501 synthesis were inserted into pLYZL102. The resulted plasmid pLYhemA2D was electrotransported to A. orientalis and the single cross-over transformants were obtained. Then pLYZL104 was introduced into the single cross-over strain and the efficiency of the homologous recombination was analyzedl before and after the sceS expression. Subsequently, the fermentation products of AhemA2 mutants were assayed by HPLC. Results Expression of sceS gene in A. orientalis resulted in promotion of homologous recombination and markerless deletion of hemA2. Conclusion I-SceI toolkit was useful for markless genetic manipulation in A. orientalis, and hemA2 was one of key genes for ECO-0501 synthesis.

关 键 词：I-SceI内切酶 东方拟无枝酸菌 ECO-0501 hemA2 

分 类 号：R978.1[医药卫生—药品]