大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用

Characterization of Promoters in the Glycolytic Pathway and Tricarboxylic Acid Cycle of E.coli and Its Application

启动子是控制基因转录的重要元件,也是合成生物学研究和细胞工厂设计的关键环节。糖酵解途径和三羧酸循环是糖类分解代谢的中心代谢,受到包括启动子强度在内的严格调控。为了筛选一系列能满足合成生物学研究和细胞工厂设计需要的不同强度的内源性组成型启动子,利用报告基因——红色荧光蛋白m Cherry和在线分析软件,系统研究了大肠杆菌糖酵解和三羧酸循环中27个启动子的强度和核心结构元件。结果表明:这些启动子的强度范围变化很大,最强启动子Pgap A的强度是最弱启动子Pacn A强度的43. 6倍;启动子的-10序列和-35序列与它们的一致序列也不完全相同,两者之间的距离为17±3bp;但是,启动子的强度和启动子的结构特征基本一致。应用最强启动子Pgap A在重组大肠杆菌DH5αΔpck中分别表达磷酸烯醇式丙酮酸羧化酶基因和丙酮酸激酶基因,它们的酶活性分别提高了0. 32和1. 57倍,柠檬酸产量也提高了124. 7%和75. 5%。这些不同强度的启动子为大肠杆菌的合成生物学研究和细胞工厂设计奠定了一定的基础。

The promoters are important components that regulate gene transcription,and key parts in synthetic biology research and cell factory design.Glycolytic pathway and tricarboxylic acid(TCA)cycle are the central metabolisms of carbohydrate catabolism and are strictly regulated by promoter strength.In order to screen some endogenous constitutive promoters with various strength necessary for synthetic biology studies and cell factory design of Escherichia coli,the strength and core structural elements of 27 promoters in glycolytic pathway and TCA cycle of E.coli were systematically studied using the red fluorescent protein(RFP)mCherry as the reporter gene and online analysis software.The results showed that the strength range of these promoters varied greatly,and the strength of the strongest promoter PgapA was 43.6 times that of the weakest promoter PacnA.Moreover,the-10 and-35 sequences of promoters are not exactly same as their consistent sequences,and the spacer between them is 17±3 bp.However,the strength of the promoters was basically consistent with the structural characteristics of the promoters.Using the strongest promoter PgapA,Phosphoenolpyruvate carboxylase and pyruvate kinase were expressed in recombinant E.coli DH5αΔpck,respectively.Their enzyme activity was increased by 0.32 and 1.57 times,respectively,and the production of citric acid was also increased by 124.7%and 75.5%.These promoters with different strength have laid a foundation for the study of synthetic biology and the design of cell factory of E.coli.