Data_Sheet_1_Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species.ZIP

Cyanobacteria utilize sunlight to convert carbon dioxide into a wide variety of secondary metabolites and show great potential for green biotechnology applications. Although cyanobacterial synthetic biology is less mature than for other heterotrophic model organisms, there are now a range of molecular tools available to modulate and control gene expression. One area of gene regulation that still lags behind other model organisms is the modulation of gene transcription, particularly transcription termination. A vast number of intrinsic transcription terminators are now available in heterotrophs, but only a small number have been investigated in cyanobacteria. As artificial gene expression systems become larger and more complex, with short stretches of DNA harboring strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regions from unwanted interference is becoming more important. In this study, we adapted a dual reporter tool for use with the CyanoGate MoClo Assembly system that can quantify and compare the efficiency of terminator sequences within and between different species. We characterized 34 intrinsic terminators in Escherichia coli, Synechocystis sp. PCC 6803, and Synechococcus elongatus UTEX 2973 and observed significant differences in termination efficiencies. However, we also identified five terminators with termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic species and cyanobacteria.

蓝细菌（Cyanobacteria）可利用光能将二氧化碳转化为多种次生代谢产物，在绿色生物技术应用领域展现出巨大潜力。尽管蓝细菌合成生物学的成熟度仍不及其他异养模式生物，但目前已具备一系列可用于调控基因表达的分子工具。然而，基因转录调控（尤其是转录终止）仍是当前蓝细菌基因调控领域落后于其他模式生物的方向之一。目前异养生物中已拥有大量内源转录终止子（intrinsic transcription terminators），但蓝细菌中仅对少数这类终止子开展了研究。随着人工基因表达系统日趋大型化、复杂化，短DNA片段可搭载强启动子与多基因表达盒，因此高效终止转录、隔绝下游区域免受非预期干扰的需求愈发迫切。本研究适配了一套可用于CyanoGate MoClo组装系统的双报告基因工具，能够定量比较不同物种内部及跨物种的终止子序列效率。我们在大肠杆菌（Escherichia coli）、集胞藻PCC 6803（Synechocystis sp. PCC 6803）以及长聚球藻UTEX 2973（Synechococcus elongatus UTEX 2973）中对34个内源转录终止子进行了功能表征，并观察到不同终止子的终止效率存在显著差异。此外，我们还鉴定出5个在三个受试物种中终止效率均超过96%的终止子，这表明部分内源转录终止子在异养生物与蓝细菌中均可保持稳定的功能表现。