Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes
Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes
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DOI：
10.1016/j.ymben.2013.07.006
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发表时间：
2013-09-01
影响因子：
8.4
通讯作者：
Luzhetskyy, Andriy
中科院分区：
文献类型：
--
作者：
Siegl, Theresa;Tokovenko, Bogdan;Luzhetskyy, Andriy
我们基于广泛使用的 ermEp1 启动子组成型启动子的 10 和 35 个共有序列，开发了放线菌合成启动子文库。使用简并引物将位于这些共有序列的上游、中间和下游的序列随机化，并克隆到gusA报告基因上游的整合质粒中。使用该系统，我们创建了与 ermEp1 相比强度范围从 2% 到 319% 的启动子。最强的合成启动子用于原理验证方法，以实现天然 III 型聚酮合酶的过表达。我们观察到gusA报告基因RNA-Seq读数的数量与GusA报告蛋白活性之间存在高度相关性，表明GusA确实是一个转录水平的报告系统。 (C) 2013 Elsevier Inc. 保留所有权利。
We developed a synthetic promoter library for actinomycetes based on the 10 and 35 consensus sequences of the constitutive and widely used ermEp1 promoter. The sequences located upstream, in between and downstream of these consensus sequences were randomised using degenerate primers and cloned into an integrative plasmid upstream of the gusA reporter gene. Using this system, we created promoters with strengths ranging from 2% to 319% compared with ermEp1. The strongest synthetic promoter was used in a proof-of-principle approach to achieve the overexpression of a natural type III polyketide synthase. We observed high correlation between the number of gusA reporter gene RNA-Seq reads and the GusA reporter protein activity, indicating that GusA is indeed a transcription-level reporter system. (C) 2013 Elsevier Inc. All rights reserved.