A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli
A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli
复制标题
DOI：
10.1016/j.jbiotec.2004.03.010
复制
发表时间：
2004-07-01
影响因子：
4.1
通讯作者：
Isaksson, LA
中科院分区：
文献类型：
--
作者：
Hägg, P;de Pohl, JW;Isaksson, LA
在细菌细胞分裂期间，基于质粒的表达载体向子细胞的不均匀分布导致生长期间无质粒细胞的比例增加。这是一个主要的工业问题，导致在载体携带细菌的大规模培养过程中产物产率降低和生产成本增加。出于这个原因，必须提供杀死无质粒细胞的选择。获得这种理想选择的最常规方法是在质粒中插入一些抗生素抗性基因，然后在相应抗生素的存在下培养细菌。我们在这里描述了一种宿主/质粒大肠杆菌系统，该系统具有完全稳定的质粒，可以在不使用抗生素选择的情况下维持。质粒得以保留，因为它携带大肠杆菌菌株中的小必需基因infA（编码翻译起始因子1，IF 1），该大肠杆菌菌株已缺失其染色体infA基因。因此，只有携带质粒的细胞才能生长，使菌株完全依赖于质粒的维持。因此，在培养过程中不需要基于抗生素的选择，并且在最终菌株和最终质粒中均不存在抗生素抗性基因。无质粒细胞即使在长时间的连续生长后也不会积累。在LB和确定成分培养基中，对照菌株和携带质粒的菌株的生长速率彼此没有区别。基于infA的质粒稳定性系统应消除由表达载体的损失和培养基中抗生素的使用引起的工业化培养问题。此外，消除了由抗生素和抗生素抗性基因的释放引起的环境问题，这些问题可能会在细菌种群之间产生水平基因转移。(C)2004 Elsevier B. V.保留所有权利。
Uneven distribution of plasmid-based expression vectors to daughter cells during bacterial cell division results in an increasing proportion of plasmid free cells during growth. This is a major industrial problem leading to reduction of product yields and increased production costs during large-scale cultivation of vector-carrying bacteria. For this reason, a selection must be provided that kills the plasmid free cells. The most conventional method to obtain this desired selection is to insert some gene for antibiotic resistance in the plasmid and then grow the bacteria in the presence of the corresponding antibiotic.We describe here a host/plasmid Escherichia coli system with a totally stable plasmid that can be maintained without the use of antibiotic selection. The plasmid is maintained, since it carries the small essential gene infA (coding for translation initiation factor 1, IF1) in an E coli strain that has been deleted for its chromosomal infA gene. As a result only plasmid carrying cells can grow, making the strain totally dependent on the maintenance of the plasmid. A selection based on antibiotics is thus not necessary during cultivation, and no antibiotic-resistance genes are present neither in the final strain nor in the final plasmid. Plasmid-free cells do not accumulate even after an extended period of continuous growth. Growth rates of the control and the plasmid harboring strains are indistinguishable from each other in both LB and defined media.The indicated approach can be used to modify existing production strains and plasmids to the described concept. The infA based plasmid stability system should eliminate industrial cultivation problems caused by the loss of expression vector and use of antibiotics in the cultivation medium. Also environmental problems caused by release of antibiotics and antibiotic resistance genes, that potentially can give horizontal gene transfer between bacterial populations, are eliminated. (C) 2004 Elsevier B.V. All rights reserved.