National Engineering Laboratory for Cereal Fermentation Technology, Key Laboratory of Industrial Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
ACS Synth Biol. 2021 Jan 15;10(1):38-48. doi: 10.1021/acssynbio.0c00405. Epub 2020 Dec 31.
Promoters and ribosome binding sites (RBSs) are routinely applied in gene expression regulation, but their orthogonality and combinatorial effects have not yet been systematically studied in Here, 17 core promoters and 29 RBSs in were characterized, which exhibited 470-fold and 430-fold in transcriptional and translational activity, respectively. By comparing the expression of two reporter genes regulated by multiple RBSs, the RBS efficacy showed significant dependence on the gene context, besides the RBSs' strength, reflecting the poor orthogonality of RBSs. Bicistron-modified RBS (referred as bc-RBS) was adapted to , which improved RBS reliability. By coupling a series of promoters with RBSs/bc-RBSs, a much broader regulation range that spanned 4 orders of magnitude was observed compared with that of a sole element, and the contribution to gene expression of RBS was more than that of promoter. Finally, promoters and RBSs were applied as built-in elements to fine-tune the gene cluster in the arginine synthesis pathway in . Compared with the original strain, more arginine (1.61-fold) or citrulline (2.35-fold) was accumulated in a 7 L bioreactor by strains with the gene expression regulation system rationally engineered. We demonstrated that, via combination of well-characterized gene elements, and overall consideration for both transcription and translation, the biosynthesis pathway can be effectively balanced, and the yield of a target metabolite can be further improved.
启动子和核糖体结合位点（RBS）通常用于基因表达调控，但它们在中的正交性和组合效应尚未得到系统研究。在这里，我们对 中的 17 个核心启动子和 29 个 RBS 进行了表征，它们在转录和翻译活性方面分别表现出 470 倍和 430 倍的活性。通过比较两个受多个 RBS 调控的报告基因的表达，除了 RBS 的强度外，RBS 的功效还显示出对基因背景的显著依赖性，反映了 RBS 的正交性较差。双顺反子修饰的 RBS（简称 bc-RBS）被适应于 ，这提高了 RBS 的可靠性。通过将一系列启动子与 RBSs/bc-RBSs 相结合，与单一元件相比，观察到更宽的调节范围跨越了 4 个数量级，并且 RBS 对基因表达的贡献大于启动子。最后，启动子和 RBS 被用作内置元件来微调精氨酸合成途径中的基因簇。与原始菌株相比，通过合理设计基因表达调控系统，在 7 L 生物反应器中，菌株积累的精氨酸（1.61 倍）或瓜氨酸（2.35 倍）更多。我们证明，通过组合特征良好的基因元件，并全面考虑转录和翻译，生物合成途径可以得到有效平衡，目标代谢产物的产量可以进一步提高。
