RoCi - 一个基于滚动循环复制的单步多副本集成系统
Martzel Antsotegi-Uskola1, Vasil D'Ambrosio2, Brian King Himm Mo2
1Department of Biotechnology and Biomedicine, Technical University of Denmark, Section for Synthetic Biology, 2800 Kongens Lyngby, Denmark.
Nucleic acids research
|January 14, 2026
概括
我们开发了RoCi,这是一种将真菌中的多个基因拷贝集成的新方法,增强了酶和代谢物生产. 这种滚动循环复制技术简化了用于工业应用的细胞工厂建设.
科学领域:
- 分子生物学分子生物学
- 合成生物学 合成生物学
- 生物技术是生物技术.
背景情况:
- 菌是生产酶和代谢物的关键细胞工厂.
- 产生高产的真菌菌株往往需要整合多个基因拷贝,这是一个挑战.
- 目前用于多个基因整合的现有方法通常是复杂和低效的.
研究的目的:
- 开发一种简单有效的方法,将多个基因表达录音带集成到真菌中定义的基因组位点中.
- 改进真菌细胞工厂的建设,以提高代谢物和酶的生产.
- 为了证明该方法在不同真菌物种中的适用性.
主要方法:
- 开发了RoCi (滚动圆集成),一种利用滚动圆复制进行基因盒集成的方法.
- 在不需要基于大肠杆菌的克隆的情况下执行RoCi,从而实现自动化实验.
- 在Aspergillus nidulans中应用RoCi,以产生多达70个mRFP副本的菌株.
主要成果:
- RoCi促进了多拷贝基因阵列的稳定集成,支持在固体介质中至少150代高稳定的mRFP生产.
- 通过将基因表达盒优化为有缺陷的促进器控制选择标记,在液体培养中减轻了轻微的拷贝损失.
- 与单复制品菌株相比,RoCi的应用导致cordicepin和β-glucuronidase的产量增加了约10倍.
- 在工业真菌劳动马中成功应用RoCi 黑色菌和小菌.
结论:
- RoCi是一种简单,高效和多用途的方法,用于在真菌中集成多个基因副本.
- 该方法显著提高了真菌细胞工厂生产有价值的代谢物和酶的产量.
- 对于合成生物学和工业生物技术的应用,RoCi具有前景,包括A. niger和A. oryzae.
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