Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli

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• 作者：Marcelo C. Bassalo ; Andrew D. Garst ; Andrea L. Halweg-Edwards ; William C. Grau ; Dylan W. Domaille ; Vivek K. Mutalik ; Adam P. Arkin ; Ryan T. Gill
• 刊名：ACS Synthetic Biology
• 出版年：2016
• 出版时间：July 15, 2016
• 年：2016
• 卷：5
• 期：7
• 页码：561-568
• 全文大小：423K
• 年卷期：0
• ISSN：2161-5063

文摘

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.