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Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number

Kang, Chae Won, Lim, Hyun Gyu, Yang, Jina, Noh, Myung Hyun, Seo, Sang Woo, Jung, Gyoo Yeol
Metabolic engineering 2018 v.48 pp. 121-128
Escherichia coli, antibiotics, auxotrophs, chromosomes, essential genes, gene expression, lycopene, plasmids, translation (genetics), value-added products
Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.