Engineered biosynthesis of colibactin in Escherichia coli

我们的异种生产系统的目标是大肠杆菌。大肠杆菌蛋白是一种从大肠杆菌he3034中分离出来的聚酮-非核糖体肽的杂交分子，可引起DNA双链断裂并激活DNA损伤检查点通路，导致细胞周期阻滞并最终导致细胞死亡。对大肠杆菌基因簇进行测序。大肠杆菌基因簇长55000个碱基，共编码了18个开放阅读框。该生物合成基因簇由7个启动子转录。生物合成基因组装：大肠杆菌生产大肠杆菌素，7个操纵子分别在大肠杆菌中表达。这7个操纵子被组装成3个独立的质粒，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。这种多单顺反电子排列是在大肠杆菌中异源生产6-dEB、耶尔希那巴蛋白和利福霉素前体系统的重要改进。多顺反子排列简化了组装过程，也最大限度地减少了转录过长多顺反子基因时可能的过早终止和mRNA降解。此外，作为先前系统的改进，我们使用了复制和抗生素抗性基因的正交起源，以确保所有三种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.