Park Geunyung, Yang Jina, Seo Sang Woo
Interdisciplinary Program in Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
Department of Chemical Engineering, Jeju National University, 102, Jejudaehak-ro, Jeju-si, Jeju-do, 63243, Korea.
J Biol Eng. 2024 Dec 19;18(1):71. doi: 10.1186/s13036-024-00460-1.
Manipulating the gene expression is the key strategy to optimize the metabolic flux. Not only transcription, translation, and post-translation level control, but also the dynamic plasmid copy number (PCN) control has been studied. The dynamic PCN control systems that have been developed to date are based on the understanding of origin replication mechanisms, which limits their application to specific origins of replication and requires the use of antibiotics for plasmid maintenance. In this study, we developed a dynamic PCN control system for Escherichia coli that is maintained without antibiotics. This is achieved by regulating the transcription level of the translation initiation factor IF-1 (infA), an essential gene encoded on the plasmid, while deleting it from the plasmid-bearing host cell.
When validated using GFP as a reporter protein, our system demonstrated a 22-fold dynamic range in PCN within the CloDF13 origin. The system was employed to determine the optimal copy number of the plasmid carrying the cad gene, which converts an intermediate of the tricarboxylic acid cycle (TCA cycle) to itaconic acid. By optimizing the PCN, we could achieve an itaconic acid titer of 3 g/L, which is 5.3-fold higher than the control strain.
Our system offers a strategy to identify the optimal expression level of genes that have a competitive relationship with metabolic pathways crucial for the growth of the host organism. This approach can potentially be applied to other bacterial hosts by substituting the sensing module or the essential gene.
操纵基因表达是优化代谢通量的关键策略。不仅转录、翻译和翻译后水平的调控已被研究，动态质粒拷贝数（PCN）调控也受到关注。迄今为止开发的动态PCN控制系统是基于对复制起点机制的理解，这限制了它们在特定复制起点的应用，并且需要使用抗生素来维持质粒。在本研究中，我们开发了一种用于大肠杆菌的动态PCN控制系统，该系统无需抗生素即可维持。这是通过调节质粒上编码的必需基因——翻译起始因子IF-1（infA）的转录水平，同时从携带质粒的宿主细胞中删除该基因来实现的。
当使用绿色荧光蛋白（GFP）作为报告蛋白进行验证时，我们的系统在CloDF13复制起点内的PCN显示出22倍的动态范围。该系统用于确定携带将三羧酸循环（TCA循环）中间体转化为衣康酸的cad基因的质粒的最佳拷贝数。通过优化PCN，我们可以实现3 g/L的衣康酸滴度，这比对照菌株高5.3倍。
我们的系统提供了一种策略，用于确定与宿主生物体生长至关重要的代谢途径具有竞争关系的基因的最佳表达水平。通过替换传感模块或必需基因，这种方法有可能应用于其他细菌宿主。
