Dynamic control of the plasmid copy number maintained without antibiotics in Escherichia coli.

Background: 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.

Results: 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.

Conclusions: 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.