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.

中文翻译：

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大合成途径通过λ-Red和CRISPR / Cas9依赖选择方法的同源性重组到大肠杆菌基因组中。

背景技术为了生物合成途径组装，代谢工程经常需要许多异源基因的基因组整合。尽管在模型微生物大肠杆菌的基因组编辑方面取得了很大进展，但与小DNA整合相比，将大途径整合到基因组中以稳定化学生产仍然具有挑战性。结果我们为大肠杆菌中的大型合成途径整合开发了一种λ-Red辅助同源依赖重组技术。通过这种方法，我们可以在一个步骤中将多达12 kb的DNA模块整合到大肠杆菌W3110的染色体中。该方法的效率可以达到100％，从而显着提高了集成效率，克服了采用常规方法的集成规模的局限性。此外，使用我们的系统将甲基磷酸赤藓醇4-磷酸酯酶（MEP）途径和番茄红素合成途径中的限制性步骤整合到W3110基因组中。随后，与初始菌株和参考菌株相比，最终菌株的产量分别提高了106倍和4.4倍。结论除了预先存在的方法外，我们的系统还提供了一种避免使用质粒的可选策略，以及一种在大肠杆菌中进行大型合成途径组装的有价值的工具。