...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Applied Microbiology and Biotechnology >I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei
【24h】

I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei

机译：I-SceI介导的双链DNA断裂刺激里氏木霉工业真菌中有效的基因靶向

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Delta ku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.
机译：在工业微生物中生产酶的表达盒的靶向整合是理想的，尤其是当筛选酶变体以改善酶性能时。但是，当前用于目标积分的方法效率低下，并且转换频率较低。在这项研究中，我们表达了酿酒酵母I-SceI大范围核酸酶，以在里氏木霉基因组中的指定位点产生双链断裂。我们显示，当在转化过程中添加侧翼与I-SceI着陆基因座同源的区域的外源DNA盒时，可以有效地修复I-SceI介导的双链DNA断裂。与对照转化相比，转化效率提高了约六倍。通过I-SceI介导的基因靶向获得的转化体的分析表明，约三分之二的转化体是由预定位点的同源重组事件引起的。在DNA盒整合后反选择pyrG标记丢失的转化子表明，几乎所有克隆都在预定位点含有该盒。使用定向整合的葡糖淀粉酶表达盒对独立获得的转化体进行分析表明，在转化体中葡糖淀粉酶的产量很高并且显示出有限的变异。总之，本研究开发的基因靶向系统显着提高了转化效率以及同源重组效率，并省略了Delta ku70菌株的使用。它也适用于里氏木霉中酶变体或基因库的高通量筛选。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号