Huang Ying, Ma Kang, Li Yan, Li Qingyan, Lu Fuping, Zhang Xueli, Sun Zhe
College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Synth Syst Biotechnol. 2025 Jun 22;10(4):1150-1159. doi: 10.1016/j.synbio.2025.06.008. eCollection 2025 Dec.
serves as an important platform for heterologous protein production, with the nisin-controlled gene expression (NICE) system being widely employed for regulated protein overexpression. However, the NICE system relies on the native RNA polymerase, which limits transcriptional efficiency, and there remains a lack of tools enabling continuous target gene mutagenesis in . In this study, we enhanced the NICE system by integrating the highly processive T7 RNA polymerase (T7RNAP) to boost protein expression. A theophylline-dependent riboswitch, RbxE, was incorporated into the nisin-induced promoter to mitigate the toxicity caused by basal T7RNAP expression in . Directed mutagenesis of the riboswitch region between the stem-loop and the ribosome binding site optimized T7RNAP expression, leading to a 2.4-fold increase upon nisin and theophylline induction in The resulting NICE-T7 system achieved a 2.8-fold increase in GFP compared to the original NICE system. Furthermore, adenosine deaminase TadA8e was fused to T7RNAP to generate the MutaT7LL system, facilitating targeted A-to-G mutagenesis and successfully reactivated an erythromycin resistance gene with a mutation efficiency of 1.33 × 10. Overall, this study presents an upgraded NICE system that enhances protein production and enables continuous in vivo mutagenesis of target genes in .
作为异源蛋白生产的重要平台，乳链菌肽控制的基因表达（NICE）系统被广泛用于调控蛋白的过表达。然而，NICE系统依赖于天然RNA聚合酶，这限制了转录效率，并且仍然缺乏能够在……中进行连续靶基因诱变的工具。在本研究中，我们通过整合高度持续合成的T7 RNA聚合酶（T7RNAP）来增强NICE系统，以提高蛋白表达。将一种依赖茶碱的核糖开关RbxE整合到乳链菌肽诱导型启动子中，以减轻……中基础T7RNAP表达所引起的毒性。对茎环与核糖体结合位点之间的核糖开关区域进行定向诱变优化了T7RNAP表达，在乳链菌肽和茶碱诱导下，……中的表达提高了2.4倍。与原始NICE系统相比，所得的NICE-T7系统使绿色荧光蛋白（GFP）表达提高了2.8倍。此外，将腺苷脱氨酶TadA8e与T7RNAP融合以生成MutaT7LL系统，促进了靶向A到G的诱变，并成功重新激活了一个红霉素抗性基因，诱变效率为1.33×10。总体而言，本研究提出了一种升级的NICE系统，该系统提高了蛋白产量，并能够在……中对靶基因进行连续的体内诱变。
