...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Applied Microbiology >Counterselection System for Geobacillus kaustophilus HTA426 through Disruption of pyrF and pyrR
【24h】

Counterselection System for Geobacillus kaustophilus HTA426 through Disruption of pyrF and pyrR

机译：通过破坏pyrF和pyrR的方法对嗜碱芽孢杆菌HTA426进行反选择系统

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (Δ pyrF ) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (Δ pyrF Δ pyrR ) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF -based counterselection system in a Bacillus -related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus . Our results also suggest a new strategy for establishment of counterselection systems.
机译：反选择系统通过实现对将标记基因导入微生物和从微生物中消除标记的积极选择，可以促进微生物中无标记的遗传修饰。在这里，我们报告了通过同时破坏pyrF和pyrR基因而建立的嗜空芽孢杆菌HTA426的反选择系统。同源重组破坏了嘧啶生物合成和5-氟乳清酸（5-FOA）代谢为有毒代谢产物所必需的pyrF基因。所得的MK54菌株（ΔpyrF）对尿嘧啶是营养缺陷的，并且对5-FOA具有抗性。补充有pyrF的MK54是尿嘧啶的原养型，但在存在尿嘧啶的情况下对5-FOA不敏感。为了赋予5-FOA敏感性，编码了通过检测尿嘧啶衍生物来抑制嘧啶生物合成的衰减器的pyrR基因被破坏。所得的MK72菌株（ΔpyrFΔpyrR）对尿嘧啶是营养缺陷的，并且对5-FOA具有抗性。即使在存在尿嘧啶的情况下，补充有pyrF的MK72对尿嘧啶和5-FOA也敏感。结果表明，pyrF可以作为MK72中的反选择标记，异源β-半乳糖苷酶和α-淀粉酶基因的有效无标记整合证明了这一点。整合的基因在嗜碱链球菌中功能性表达，并赋予嗜热菌新功能。这份报告描述了在芽孢杆菌相关细菌中首次建立基于pyrF的反选择系统，以及首次展示了嗜酒假单胞菌中的同源重组和异源基因表达。我们的结果还提出了建立反选择系统的新策略。

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