Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.
Nucleic Acids Res. 2022 Apr 22;50(7):4171-4186. doi: 10.1093/nar/gkac206.
As the design of genetic circuitry for synthetic biology becomes more sophisticated, diverse regulatory bioparts are required. Despite their importance, well-characterized 3'-untranslated region (3'-UTR) bioparts are limited. Thus, transcript 3'-ends require further investigation to understand the underlying regulatory role and applications of the 3'-UTR. Here, we revisited the use of Term-Seq in the Escherichia coli strain K-12 MG1655 to enhance our understanding of 3'-UTR regulatory functions and to provide a diverse collection of tunable 3'-UTR bioparts with a wide termination strength range. Comprehensive analysis of 1,629 transcript 3'-end positions revealed multiple 3'-termini classes generated through transcription termination and RNA processing. The examination of individual Rho-independent terminators revealed a reduction in downstream gene expression over a wide range, which led to the design of novel synthetic metabolic valves that control metabolic fluxes in branched pathways. These synthetic metabolic valves determine the optimal balance of heterologous pathways for maximum target biochemical productivity. The regulatory strategy using 3'-UTR bioparts is advantageous over promoter- or 5'-UTR-based transcriptional control as it modulates gene expression at transcription levels without trans-acting element requirements (e.g. transcription factors). Our results provide a foundational platform for 3'-UTR engineering in synthetic biology applications.
随着合成生物学中基因电路设计变得更加复杂，需要更多不同的调控生物部件。尽管它们很重要，但经过良好表征的 3'-非翻译区（3'-UTR）生物部件是有限的。因此，需要进一步研究转录本 3'-末端，以了解 3'-UTR 的潜在调控作用和应用。在这里，我们重新使用了 Term-Seq 在大肠杆菌菌株 K-12 MG1655 中，以增强我们对 3'-UTR 调控功能的理解，并提供了广泛的可调 3'-UTR 生物部件，具有广泛的终止强度范围。对 1629 个转录本 3'-末端位置的综合分析揭示了通过转录终止和 RNA 处理产生的多个 3'-末端类。对单个 Rho 非依赖性终止子的研究表明，在广泛的范围内，下游基因表达减少，这导致了设计新型合成代谢阀，以控制分支途径中的代谢通量。这些合成代谢阀确定了异源途径的最佳平衡，以实现最大目标生化产物产量。与基于启动子或 5'-UTR 的转录控制相比，使用 3'-UTR 生物部件的调控策略具有优势，因为它可以在不依赖反式作用元件（例如转录因子）的情况下在转录水平上调节基因表达。我们的研究结果为合成生物学应用中的 3'-UTR 工程提供了一个基础平台。
