Department Microbial Natural Products, Actinobacteria Metabolic Engineering Group, Saarland University, Saarbrücken, Germany.
Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) Helmholtz Center for Infectious Research (HZI), Campus C2.3, 66123, Saarbrücken, Germany.
Sci Rep. 2018 Jan 11;8(1):491. doi: 10.1038/s41598-017-18846-1.
The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routinely, their translation and termination efficiencies have not been systematically studied in Actinobacteria. Here, we analyzed the influence of the regions surrounding RBSs on gene expression in these bacteria. We demonstrated that inappropriate RBSs can reduce the expression efficiency of a gene to zero. We developed a genetic device - an in vivo RBS-selector - that allows selection of an optimal RBS for any gene of interest, enabling rational control of the protein expression level. In addition, a genetic tool that provides the opportunity for measurement of termination efficiency was developed. Using this tool, we found strong terminators that lead to a 17-100-fold reduction in downstream expression and are characterized by sufficient sequence diversity to reduce homologous recombination when used with other elements. For the first time, a C-terminal degradation tag was employed for the control of protein stability in Streptomyces. Finally, we describe a collection of regulatory elements that can be used to control metabolic pathways in Actinobacteria.
次生代谢基因簇的设计和工程以复杂的遗传组织为特征，需要开发可重复使用的经过良好表征的遗传调控元件集合。尽管经常使用一些内在的终止子和 RBS，但它们在放线菌中的翻译和终止效率尚未得到系统研究。在这里，我们分析了 RBS 周围区域对这些细菌中基因表达的影响。我们证明了不合适的 RBS 会使基因的表达效率降低到零。我们开发了一种遗传装置——体内 RBS 选择器，它允许为任何感兴趣的基因选择最佳的 RBS，从而实现对蛋白质表达水平的合理控制。此外，还开发了一种提供测量终止效率机会的遗传工具。使用该工具，我们发现了强大的终止子，可使下游表达降低 17-100 倍，并且具有足够的序列多样性，可在与其他元件一起使用时减少同源重组。我们首次将 C 末端降解标签用于控制链霉菌中的蛋白质稳定性。最后，我们描述了一组调控元件，可用于控制放线菌中的代谢途径。
