Promoter engineering has emerged as a powerful tool to activate transcriptionally silent natural product biosynthetic gene clusters found in bacterial genomes. Since biosynthetic gene clusters are composed of multiple operons, their promoter engineering requires the use of a set of regulatory sequences with a similar level of activities. Although several successful examples of promoter engineering have been reported, its widespread use has been limited due to the lack of a library of regulatory sequences suitable for use in promoter engineering of large, multiple operon-containing biosynthetic gene clusters. Here, we present the construction of a library of constitutively active, synthetic Streptomyces regulatory sequences. The promoter assay system has been developed using a single-module nonribosomal peptide synthetase that produces the peptide blue pigment indigoidine, allowing for the rapid screening of a large pool of regulatory sequences. The highly randomized regulatory sequences in both promoter and ribosome binding site regions were screened for their ability to produce the blue pigment, and they are classified into the strong, medium, and weak regulatory sequences based on the strength of a blue color. We demonstrated the utility of our synthetic regulatory sequences for promoter engineering of natural product biosynthetic gene clusters using the actinorhodin gene cluster as a model cluster. We believe that the set of Streptomyces regulatory sequences we report here will facilitate the discovery of new natural products from silent, cryptic biosynthetic gene clusters found in sequenced Streptomyces genomes.

中文翻译：

---

用于天然产物生物合成基因簇启动子工程的合成链霉菌调控序列文库

启动子工程已成为激活细菌基因组中沉默的转录天然产物生物合成基因簇的强大工具。由于生物合成基因簇由多个操纵子组成，因此其启动子工程需要使用一组具有相似活性水平的调控序列。尽管已经报道了启动子工程的几个成功的例子，但是由于缺乏适用于大型，含多个操纵子的生物合成基因簇的启动子工程的调控序列文库，其广泛使用受到了限制。在这里，我们介绍了组成型活性，合成链霉菌的文库的构建调节序列。使用单模块非核糖体肽合成酶开发了启动子测定系统，该合成酶可产生肽蓝色素靛蓝，可快速筛选大量调控序列。筛选启动子和核糖体结合位点区域中高度随机的调节序列产生蓝色色素的能力，并根据蓝色的强度将其分为强，中和弱调节序列。我们证明了我们的合成调控序列对于使用肌动球蛋白基因簇作为模型簇的天然产物生物合成基因簇的启动子工程的实用性。我们认为，链霉菌我们在此报告的调控序列将有助于从测序的链霉菌基因组中发现的沉默的，隐秘的生物合成基因簇中发现新的天然产物。