权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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。 Colibactin 是从大肠杆菌 IHE3034 中分离出的聚酮化合物-非核糖体肽的杂合分子，可引起 DNA 双链断裂并激活 DNA 损伤检查点途径，导致细胞周期停滞并最终导致细胞死亡。对大肠杆菌基因簇进行了测序。 colibactinggene簇跨越55kb长，簇内编码了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：为了生产大肠杆菌素，七个操纵子中的每一个在大肠杆菌中单独表达，确认表达。七个操纵子被组装成三个独立的质粒，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。多单顺反子排列是对之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的系统的重要改进。多单顺反子排列简化了组装过程，并最大限度地减少了转录过长多顺反子基因时潜在的过早终止和 mRNA 降解。此外，作为对先前系统的改进，我们使用正交复制起点和抗生素抗性基因来确保所有三种质粒在大肠杆菌中的稳定保留。