Exploitation of a Type 1 Toxin-Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium limosum.
Exploitation of a Type 1 Toxin-Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium limosum.
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DOI：
10.3390/microorganisms11051256
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发表时间：
2023-05-10
期刊：
影响因子：
4.5
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中科院分区：
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此前已使用基于 CRISPR 的诱变方法获得了厌氧甲基营养菌真杆菌中的靶向突变。在这项研究中，来自卡兰德里真杆菌的RelB家族毒素被置于脱水四环素敏感启动子的控制下，形成诱导型反选择系统。该诱导系统与非复制整合诱变载体相结合，在 Limosum B2 真杆菌中产生精确的基因删除。本研究的目标基因是编码组氨酸生物合成基因 hisI、甲醇甲基转移酶和类咕啉蛋白 mtaA 和 mtaC 以及 mtcB（编码 Mttb 家族甲基转移酶，该酶先前已被证明可以使左旋肉碱去甲基化）。 hisI 内的定向删除导致了预期的组氨酸营养缺陷型，而 mtaA 和 mtaC 的删除都消除了对甲醇的自养生长。 mtcB 的缺失被证明可以消除 L-肉碱上的 E. limosum 的生长。在分离转化体菌落的初始选择步骤之后，仅需要单个诱导步骤即可获得所需靶标的突变菌落。诱导型反选择标记和非复制整合质粒的组合允许对 E. limosum 进行快速基因编辑。
Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.