Lobanova Juliya S, Gorshkova Natalya V, Krylov Alexander A, Stoynova Nataliya V, Mashko Sergey V
Ajinomoto-Genetika Research Institute, 117545 Moscow, Russian Federation.
Ajinomoto-Genetika Research Institute, 117545 Moscow, Russian Federation.
J Microbiol Methods. 2022 Sep;200:106555. doi: 10.1016/j.mimet.2022.106555. Epub 2022 Aug 6.
A novel genome editing method for repeated introduction of foreign DNA, including insertion of rather large DNA fragments, into predesigned points in the Corynebacterium glutamicum chromosome was developed. The method is based on the implementation of the Dual-In/Out strategy, which was previously provided in Escherichia coli according to recombineering-based methods (Minaeva et al., 2008) and allowed step-by-step construction of marker-less plasmid free recombinant strains. The strategy, suggested in the current study, is based on (i) E. coli Rac prophage RecE/RecT-dependent recombineering; (ii) corynephage ϕ16 (Int/Xis)- and E. coli phage P1 Cre-mediated site-specific recombination systems; and (iii) the development of a C. glutamicum electrotransformation protocol with donor chromosomal DNA for combining of obtained modifications. It was found, that for each tested C. glutamicums strain, the efficiency of the different modifications for electrotransformation fluctuated significantly (up to two orders of magnitude), likely due to the recombinogenic accessibility of the corresponding locus of the bacterial chromosome. To avoid this difficulty, we proposed the phage Mu-driven transposition as a powerful approach for pre-selection of chromosomal regions convenient for single insertions and their further combination in a one strain. Additionally, it was found that the expression of RecE/RecT coding genes in the recipient strain facilitated the inheritance of the penetrated DNA. It is proposed that the developed strategy in general and its separate elements should be helpful for broadening the genetic toolbox needed for genome editing of targeted C. glutamicum strains.
开发了一种新的基因组编辑方法，用于将外源DNA（包括相当大的DNA片段）重复引入谷氨酸棒杆菌染色体的预先设计位点。该方法基于双进双出策略的实施，该策略先前是根据基于重组工程的方法在大肠杆菌中提出的（米娜耶娃等人，2008年），并允许逐步构建无标记的无质粒重组菌株。本研究中提出的策略基于：（i）大肠杆菌Rac原噬菌体RecE/RecT依赖性重组工程；（ii）棒状噬菌体ϕ16（Int/Xis）和大肠杆菌噬菌体P1 Cre介导的位点特异性重组系统；（iii）开发用于结合获得的修饰的供体染色体DNA的谷氨酸棒杆菌电转化方案。结果发现，对于每个测试的谷氨酸棒杆菌菌株，电转化的不同修饰效率波动很大（高达两个数量级），这可能是由于细菌染色体相应位点的重组可及性。为了避免这一困难，我们提出噬菌体Mu驱动的转座作为一种强大的方法，用于预先选择便于单插入的染色体区域，并在一个菌株中进一步组合它们。此外，还发现受体菌株中RecE/RecT编码基因的表达促进了穿透DNA的遗传。建议总体上所开发的策略及其各个要素应有助于拓宽靶向谷氨酸棒杆菌菌株基因组编辑所需的遗传工具箱。
