Engineered biosynthesis of colibactin in Escherichia coli

我们的异源生产系统的目标是Colibactin。Colibactin是从E.coliIHE3034中分离到的一种聚酮-非核糖体肽的杂交分子，可引起DNA双链断裂和DNA损伤检查点通路的激活，导致细胞周期停滞，最终导致细胞死亡。对大肠杆菌簇簇进行了测序。该基因簇全长55kb，编码18个开放阅读框，由7个启动子组成。生物合成基因的组装：为了生产大肠杆菌，七个操纵子中的每一个都在大肠杆菌中单独表达，以确认表达。这7个操纵子被组装成3个单独的质粒，每个操纵子携带自己的T7启动子、核糖体结合位点和T7转录终止子。多单顺反子的排列是对以前在大肠杆菌中异源生产6-deb、耶尔尼阿巴菌素和利福霉素前体的系统的重要改进。多个单顺反子的排列简化了组装过程，也最大限度地减少了转录过长的多顺反子基因时可能的提前终止和mRNA降解。此外，作为对以往系统的改进，我们使用了复制基因和抗生素耐药基因的正交起始点，以确保所有三个质粒在大肠杆菌中的稳定保留。

The target of our heterologous production system is colibactin. Colibactin is a hybrid molecule of a polyketide-nonribosomal peptideisolated from Escherichia coliIHE3034, that causes DNA double-strand breaks and activation of the DNA damage checkpoint pathway, leading to cell cycle arrest and eventually to cell death. The colibactingene cluster was sequenced. The colibactingene cluster spanned 55-kilobases-long and eighteen open-reading frames were ecoded in the cluster.The biosynthetic gene cluster wastranscribed by seven promoters. Assembly of biosynthesis genes: For the production of colibactinin E. coli, each of the seven operons wasindividually expressed in E. colito confirm expression. The seven operonswere assembled into three separate plasmids with each operoncarrying its own T7 promoter, ribosomal binding site and T7 transcriptional terminator. The multimonocistronic arrangement was an important improvement from previous systems for the heterologous production of 6-dEB, yersiniabactin and rifamycin precursor in E. coli. The multimonocistronic arrangement simplify the assembly process and also minimize the potential premature terminations and mRNA degradation in transcribing excessively long polycistronic gene. In addition as an improvement of previous systems we used orthogonal origins of replication and antibiotic resistance genes to ensure the stable retention of all three plasmids in E. coli.