Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number
Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number
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DOI：
10.1016/j.ymben.2018.05.020
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发表时间：
2018-07-01
影响因子：
8.4
通讯作者：
Jung, Gyoo Yeol
中科院分区：
文献类型：
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作者：
Kang, Chae Won;Lim, Hyun Gyu;Jung, Gyoo Yeol
尽管基于质粒的表达系统在基因的多拷贝表达方面具有优势，但即使在添加抗生素的情况下，单个细胞中的质粒拷贝数(PCN)的异质性也是不可避免的。在这里，我们开发了一种合成的营养缺乏症系统，用于在不添加抗生素的情况下稳定和可调地维持大肠杆菌中的PCN。这种营养缺陷型细胞在质粒上而不是在染色体上表达INFA，这是编码翻译起始因子的重要基因之一。利用该系统，在不含抗生素的条件下，基因表达稳定地保持了40代，细胞间的差异最小。此外，改变INFA的表达水平使我们能够合理地将PCN调节5.6倍以上。这种不含抗生素的PCN控制系统与基于抗生素的传统系统相比，显著提高了衣康酸和番茄红素的产量(2倍)。总体而言，制定的战略可以成为生产无抗生素种植中增值产品的平台。
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.