Linder Marten, Haak Markus, Botes Angela, Kalinowski Jörn, Rückert Christian
CeBiTec Bielefeld, Technology Platform Genomics, Bielefeld University, Bielefeld, Germany.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States.
Front Bioeng Biotechnol. 2021 Dec 15;9:751334. doi: 10.3389/fbioe.2021.751334. eCollection 2021.
Mobile genetic elements (MGEs) contribute to instability of the host genome and plasmids. Previously, removal of the prophages in the industrial amino acid producer ATCC 13 032 resulted in strain MB001 which showed better survival under stress conditions and increased transformability. Still, eight families of Insertion Sequence (IS) elements with 27 potentially active members remain in MB001, two of which were demonstrated to be detrimental in biotechnological processes. In this study, systematical deletion of all complete IS elements in MB001 resulted in the MGE-free strain CR101. CR101 shows growth characteristics identical to the wildtype and the increased transformability of MB001. Due to its improved genome stability, we consider this strain to be an optimal host for basic research and biotechnology. As a "zero-background" host, it is also an ideal basis to study IS elements. Re-sequencing of CR101 revealed that only five spontaneous point mutations had occurred during the construction process, highlighting the low mutation rate of on the nucleotide level. In a second step, we developed an easily applicable IS-based transposon mutagenesis system to randomly transpose a selectable marker. For optimal plasmid stability during cloning in , the system utilizes a genetic switch based on the phage integrase Bxb1. Use of this integrase revealed the presence of a functional site in the genome. To avoid cross-talk with our system and increase ease-of-use, we removed the site and also inserted the Bxb1 encoding gene into the chromosome of CR101. Successful insertion of single markers was verified by sequencing randomly selected mutants. Sequencing pooled mutant libraries revealed only a weak target site specificity, seemingly random distribution of insertion sites and no general strand bias. The resulting strain, ML103, together with plasmid pML10 provides a easily customizable system for random mutagenesis in an otherwise genomically stable . Taken together, the MGE-free strain CR101, the derivative ML103, and the plasmid pML10 provide a useful set of tools to study in the future.
移动遗传元件（MGEs）会导致宿主基因组和质粒的不稳定。此前，在工业氨基酸生产菌ATCC 13032中去除原噬菌体后得到了菌株MB001，该菌株在应激条件下表现出更好的存活率且转化能力增强。尽管如此，MB001中仍保留了8个插入序列（IS）元件家族，其中27个可能具有活性，其中两个已被证明在生物技术过程中具有有害性。在本研究中，对MB001中所有完整的IS元件进行系统删除，得到了无MGEs的菌株CR101。CR101表现出与野生型相同的生长特性以及MB001增强的转化能力。由于其基因组稳定性得到改善，我们认为该菌株是基础研究和生物技术的理想宿主。作为“零背景”宿主，它也是研究IS元件的理想基础。对CR101的重新测序显示，在构建过程中仅发生了5个自发点突变，突出了核苷酸水平上的低突变率。第二步，我们开发了一种易于应用的基于IS的转座子诱变系统，用于随机转座一个选择标记。为了在克隆过程中实现最佳的质粒稳定性，该系统利用了基于噬菌体整合酶Bxb1的遗传开关。使用这种整合酶揭示了该基因组中存在一个功能性位点。为了避免与我们的系统发生串扰并提高易用性，我们去除了该位点，并将编码Bxb1的基因插入到CR101的染色体中。通过对随机选择的突变体进行测序验证了单个标记的成功插入。对混合突变体文库的测序显示，靶位点特异性较弱，插入位点似乎随机分布且无普遍的链偏向性。所得菌株ML103与质粒pML10一起为在基因组稳定的情况下进行随机诱变提供了一个易于定制的系统。综上所述，无MGEs的菌株CR101、衍生物ML103和质粒pML10为未来研究提供了一套有用的工具。
