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开发一种重组系统,以Cas9对抗选择为无标记基因组工程的乙菌Eubacterium limosum

Patrick A Sanford¹, Benjamin M Woolston¹

¹Department of Chemical Engineering, Northeastern University, 360 Huntington Avenue, 223 Cullinane, Boston, Massachusetts 02115, United States.

ACS synthetic biology

|July 21, 2024

概括

我们为Eubacterium limosum开发了一种新的基因工程系统,可以进行精确的基因组修改. 这一突破促进了其在可持续生物燃料生产和理解肠道健康方面的应用.

科学领域:

微生物学微生物学
合成生物学合成生物学
代谢工程是代谢工程.

背景情况:

利莫菌 (Eubacterium limosum) 是一个Clostridial的乙原体,有可能用于基于二氧化碳的生物燃料生产和研究肠道微生物群.
在E. limosum中,基因工程受到缺乏快速和精确的基因组修改工具的限制.

研究的目的:

开发一种高效且无痕迹的方法,用于精确地对E. limosum进行基因组修改.
为了克服E. limosum合成生物学和代谢工程中的一个关键瓶.

主要方法:

选重组酶蛋白质库以确定有效的病毒重组酶RecT用于基于寡核酸的重组.
优化系统以实现高效的单链DNA (ssDNA) 重组.
实施Cas9反选择系统以消除未重组的细胞,实现无痕和无标记的基因组点突变.

主要成果:

为E. limosum开发了一种高效的基于寡核酸的重组系统,高达2%的ssDNA重组效率.
使用Cas9反选择系统实现了高达100%的可活细胞编码所需突变.
成功创建了一个缺少细胞外聚合物质 (EPS) 基因集群的淘汰菌株,证明了该系统的实用性.

结论:

开发的系统能够在E. limosum中进行快速,简单和无痕的基因组修改,克服了重大工程挑战.
这种方法是多功能性的,可以重新定位到各种基因组位置.
提供了一种有价值的工具和等离子体库,用于推进E. limosum和其他Clostridia的基因工程.

关键词:

C1代谢过程中的代谢密闭的代谢工程密闭的代谢工程发酵气体发酵的气体是什么基因组工程是基因组工程.我们的肠道微生物组.它们是重组组合的.

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