Zhou Libang, Ren Jie, Li Zhidong, Nie Jinglei, Wang Chuang, Zeng An-Ping
Beijing Advanced Innovation Center for Soft Matter Science and Engineering , Beijing University of Chemical Technology , North Third Ring Road 15 , Chaoyang District, Beijing 100029 , China.
College of Food Science and Technology , Nanjing Agricultural University , Weigang 1 , Nanjing 210095 , PR China.
ACS Synth Biol. 2019 Oct 18;8(10):2327-2335. doi: 10.1021/acssynbio.9b00137. Epub 2019 Oct 10.
A riboswitch, a regulatory RNA that controls gene expression by specifically binding a ligand, is an attractive genetic element for the control of conditional gene expression and metabolic pathways. In this study, we identified a glycine riboswitch located in the 5'-untranslated regions of a glycine:proton symporter gene in . The glycine riboswitch is shown to contain two tandem aptamers and to function as an activator of expression of genes fused to its expression platform. Results of singlet aptamer experiments indicated that aptamer-2 has a much higher impact on regulating gene expression than aptamer-1. Further, we successfully obtained synthetic glycine-OFF riboswitches using a dual selection approach, and one of them repressed gene expression up to 10.2-fold with an improved dynamic range. The specific glycine-OFF riboswitch can function as an independent repressor in the presence of glycine, and its repression mechanism is inferred from predicted secondary structure. The selected glycine-OFF riboswitch was used to dynamically control the biosynthesis of 5-aminolevulinic acid (5-ALA) in with an unnatural 5-ALA synthetic pathway, in which glycine plays a key role. It is demonstrated that the use of a synthetic glycine-OFF riboswitch can lead to a significant increase (11%) of 5-ALA in harboring an unnatural biosynthetic pathway.
核糖开关是一种通过特异性结合配体来控制基因表达的调控RNA，是用于控制条件性基因表达和代谢途径的一种有吸引力的遗传元件。在本研究中，我们在[具体物种]的甘氨酸：质子同向转运体基因的5'-非翻译区鉴定出一个甘氨酸核糖开关。该甘氨酸核糖开关显示含有两个串联适体，并作为与其表达平台融合的基因表达的激活剂发挥作用。单适体实验结果表明，适体-2对基因表达调控的影响远高于适体-1。此外，我们使用双重筛选方法成功获得了合成的甘氨酸-OFF核糖开关，其中一个能将基因表达抑制高达10.2倍，且动态范围有所改善。特定的甘氨酸-OFF核糖开关在甘氨酸存在时可作为独立的阻遏物发挥作用，其阻遏机制可从预测的二级结构推断得出。所筛选的甘氨酸-OFF核糖开关用于通过一条非天然的5-氨基乙酰丙酸（5-ALA）合成途径动态控制[具体物种]中5-氨基乙酰丙酸的生物合成，在该途径中甘氨酸起关键作用。结果表明，使用合成的甘氨酸-OFF核糖开关可使具有非天然生物合成途径的[具体物种]中的5-ALA显著增加（11%）。
