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Background  Metabolic 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.    Results  We 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.    Conclusions  In 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 .

Homology-dependent recombination of large synthetic pathways into E. coli genome via λ-Red and CRISPR/Cas9 dependent selection methodology