Design of Ribosome Binding Sites in Streptomyces coelicolor
Design of Ribosome Binding Sites in Streptomyces coelicolor
复制标题
天蓝色链霉菌核糖体结合位点的设计
DOI：
10.2174/1570164614666170724120325
复制
发表时间：
2017-01-01
影响因子：
0.8
通讯作者：
Jiang, Hui
中科院分区：
文献类型：
--
作者：
Luo, Hong-Dou;Tao, Yang;Jiang, Hui
背景：原核生物中含有Shine-Dalgarno(SD)序列的核糖体结合位点(RBS)在核糖体鉴定中起着重要作用。目的：本研究旨在通过改造RBS来提高原核生物翻译水平下的目的蛋白产量。方法：在天蓝色链霉菌M145、Sco-RBS*和Sco-RBS0中设计了两个RBS，其中Sco-RBS*的SD序列与16S RNA的3‘端完全互补，Sco-RBS0的SD序列与16S RNA的3’端完全不互补。以增强型绿色荧光蛋白(EGFP)为报告基因，在Sco-RBS*、Sco-RBS0和天然RBS Sco-RBSactI的调控下，在天蓝色链霉菌中转录和翻译其基因。结果：用Sco-RBS*取代Sco-RBSactI后，绿色荧光蛋白的产量和绿色荧光蛋白/绿色荧光蛋白的比例分别增加2.67倍和6.07倍。用Sco-RBS0取代Sco-RBSactI后，EGFP表达水平和EGFP/EGFP mRNA比值分别下降了4.35倍和2.18倍。结论：我们提供了一种在翻译水平上提高链霉菌蛋白质产量的方法。
Background: The ribosome binding site (RBS) containing Shine-Dalgarno (SD) sequence in prokaryotes plays an important role in identification of the translation initiation site within mRNA by ribosome.Objective: Our study aimed to improve the target protein production under translational level in prokaryotes by engineering of RBSs.Method: Two RBSs were designed in Streptomyces coelicolor M145, Sco-RBS* with an SD sequence which is completely complementary to 3'-end of 16S RNA and Sco-RBS0 with an SD sequence which is completely non-complementary to 3'-end of 16S RNA. The enhanced green fluorescent protein (EGFP) was used as a reporter, whose gene was transcribed and translated under the control of Sco-RBS*, Sco-RBS0, and a native RBS Sco-RBSactI in recombinant S. coelicolor strains.Results: Replacement of Sco-RBSactI with Sco-RBS* resulted in increase of both EGFP production and the ratio of EGFP to EGFP mRNA by 2.67-fold and 6.07-fold, respectively. Replacement of Sco-RBSactI with Sco-RBS0 resulted in decrease of both EGFP and the ratio of EGFP to EGFP mRNA by 4.35-fold and 2.18-fold, respectively. Conclusion: We provided a method to increase protein production at the translational level in Streptomyces.