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Engineered biosynthesis of colibactin in Escherichia coli

大肠杆菌中大肠杆菌素的工程生物合成

基本信息

批准号：
23810026
负责人：
WANIBUCHI Kiyofumi
金额：
$ 2.08万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for Research Activity Start-up
财政年份：
2011
资助国家：
日本
起止时间：
2011 至 2012
项目状态：
已结题

项目摘要

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.

我们的异源生产系统的目标是大肠杆菌素。大肠杆菌素是从大肠杆菌IHE 3034中分离的聚酮-非核糖体肽的杂合分子，其引起DNA双链断裂和DNA损伤检查点途径的激活，导致细胞周期停滞并最终导致细胞死亡。对大肠杆菌素簇进行测序。大肠杆菌素基因簇全长55个碱基，编码18个开放阅读框，生物合成基因簇由7个启动子转录。生物合成基因的组装：用于大肠杆菌素E的生产。大肠杆菌中表达，并在大肠杆菌中表达。colito确认表达。将这7个操纵子分别组装成3个质粒，每个操纵子分别携带T7启动子、核糖体结合位点和T7转录终止子。多单顺反子排列是对以前在大肠杆菌中异源生产6-dEB、耶尔森菌素和利福霉素前体系统的重要改进。杆菌多单顺反子排列简化了组装过程，并且还最小化了在转录过长的多顺反子基因时潜在的过早终止和mRNA降解。此外，作为对以前系统的改进，我们使用了正交复制起点和抗生素抗性基因，以确保所有三种质粒在E.杆菌