Jang Sungho, Jang Sungyeon, Xiu Yu, Kang Taek Jin, Lee Sang-Hyeup, Koffas Mattheos A G, Jung Gyoo Yeol
Department of Chemical Engineering, Pohang University of Science and Technology , 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Korea.
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
ACS Synth Biol. 2017 Nov 17;6(11):2077-2085. doi: 10.1021/acssynbio.7b00128. Epub 2017 Aug 10.
Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist. Unlike the protein-based trans-regulating controllers, riboswitches can respond to their ligands faster and eliminate off-target effects. Here, we developed artificial riboswitches that activate gene expression in response to naringenin, an important flavonoid. RNA aptamers for naringenin were developed using SELEX and cloned upstream of a dual selectable marker gene to construct a riboswitch library. Two in vivo selection routes were applied separately to the library by supplementing naringenin at two different concentrations during enrichments to modulate the operational ranges of the riboswitches. The selected riboswitches were responsive to naringenin and activated gene expression up to 2.91-fold. Operational ranges of the riboswitches were distinguished on the basis of their selection route. Additionally, the specificity of the riboswitches was assessed, and their applicability as dynamic regulators was confirmed. Collectively, the naringenin riboswitches reported in this work will be valuable tools in metabolic engineering of microorganisms for the production of flavonoids.
由于微生物菌株生长速度快且适合大规模生产，因此被认为是生产黄酮类化合物的有前景的宿主。然而，目前重组菌株的生产力和产量仍未达到工业生产过程的要求。基因编码生物传感器已应用于生物合成途径的高通量筛选或动态调控，以提高生产有价值化学品的微生物菌株的性能。目前，针对黄酮类化合物的蛋白质传感器调节因子很少。与基于蛋白质的反式调节控制器不同，核糖开关对其配体的响应更快，并且可以消除脱靶效应。在这里，我们开发了一种人工核糖开关，它能响应重要的黄酮类化合物柚皮素激活基因表达。使用指数富集的配体系统进化技术（SELEX）开发了针对柚皮素的RNA适体，并将其克隆到双选择标记基因的上游以构建核糖开关文库。在富集过程中通过添加两种不同浓度的柚皮素来分别对文库应用两种体内筛选途径，以调节核糖开关的操作范围。筛选出的核糖开关对柚皮素具有响应性，并能将基因表达激活至2.91倍。根据其筛选途径区分了核糖开关的操作范围。此外，还评估了核糖开关的特异性，并证实了它们作为动态调节因子的适用性。总的来说，这项工作中报道的柚皮素核糖开关将成为微生物代谢工程中生产黄酮类化合物的有价值工具。
