Abstract
BackgroundMetabolic engineering frequently needs genomic integration of many heterologous genes for biosynthetic pathway assembly. Despite great progresses in genome editing for the model microorganism Escherichia coli, the integration of large pathway into genome for stabilized chemical production is still challenging compared with small DNA integration.ResultsWe have developed a λ-Red assisted homology-dependent recombination for large synthetic pathway integration in E. coli. With this approach, we can integrate as large as 12 kb DNA module into the chromosome of E. coli W3110 in a single step. The efficiency of this method can reach 100%, thus markedly improve the integration efficiency and overcome the limitation of the integration size adopted the common method. Furthermore, the limiting step in the methylerythritol 4-phosphate (MEP) pathway and lycopene synthetic pathway were integrated into the W3110 genome using our system. Subsequently, the yields of the final strain were increased 106 and 4.4-fold compared to the initial strain and the reference strain, respectively.ConclusionsIn addition to pre-existing method, our system presents an optional strategy for avoiding using plasmids and a valuable tool for large synthetic pathway assembly in E. coli.
Highlights
Metabolic engineering frequently needs genomic integration of many heterologous genes for biosynthetic pathway assembly
The widely used methods for genome editing in E. coli were developed on the basis of λ-Red promoted homology-dependent recombination (HDR)
When conducted the editing processes, the vector pRC-IS5 was inserted into the E. coli W3110 genome by recA-mediated HDR assisted by λ-Red
Summary
Metabolic engineering frequently needs genomic integration of many heterologous genes for biosynthetic pathway assembly. Various metabolic engineering strategies, including overexpression of key genes, The widely used methods for genome editing in E. coli were developed on the basis of λ-Red promoted homology-dependent recombination (HDR). Chromosomal integration of DNA modules with the size of about 2000 bp could be accomplished through λ-Red promoted HDR with high efficiency [4]. A 50 kb DNA module from B. subtilis 168, divided into seven segments (each ~ 6–7 kb), was inserted into E. coli genome through iterative integration [9]. This method could integrate large DNA fragment, which required many rounds of integration to achieve large synthetic pathways integration
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