Rapid and Efficient One-Step Metabolic Pathway Integration in E-coli
Rapid and Efficient One-Step Metabolic Pathway Integration in E-coli
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DOI：
10.1021/acssynbio.5b00187
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发表时间：
2016-07-01
影响因子：
4.7
通讯作者：
Gill, Ryan T.
中科院分区：
文献类型：
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作者：
Bassalo, Marcelo C.;Garst, Andrew D.;Gill, Ryan T.
将异源基因导入可遗传处理的宿主的方法是合成生物学最受欢迎的工具之一。快速、稳定地测试宿主基因组中的基因和途径的简单即插即用的构建方法将加快合成生物学和代谢工程的应用。在这里，我们描述了一种基于CRISPR的策略，该策略允许高效、单步整合大肠杆菌中的大通路。这一策略允许在广泛的同源臂大小和基因组位置范围内进行高效整合，在7个不同的基因座上的效率从70%到100%不等。为了展示大尺寸能力，我们集成了一个10kb的结构来在一天内实现异丁醇的生产。以快速和无标记的方式有效地整合整个代谢途径的能力将有助于使用大肠杆菌基因组作为稳定的测试平台来测试和设计新的途径。
Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.