Bacterial Genome Editing via a Designed Toxin–Antitoxin Cassette,ACS Synthetic Biology

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Bacterial Genome Editing via a Designed Toxin–Antitoxin Cassette
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2017-01-17 00:00:00 , DOI: 10.1021/acssynbio.6b00287
Jie Wu _{1,

2} , Aihua Deng ₁ , Qinyun Sun ₁ , Hua Bai _{1,

2} , Zhaopeng Sun ₁ , Xiuling Shang ₁ , Yun Zhang ₁ , Qian Liu ₁ , Yong Liang ₁ , Shuwen Liu ₁ , Yongsheng Che ₃ , Tingyi Wen _{1,

4}

Affiliation

Manipulating the bacterial genomes in an efficient manner is essential to biological and biotechnological research. Here, we reprogrammed the bacterial TA systems as the toxin counter-selectable cassette regulated by an antitoxin switch (TCCRAS) for genetic modifications in the extensively studied and utilized Gram-positive bacteria, B. subtilis and Corynebacterium glutamicum. In the five characterized type II TA systems, the RelBE complex can specifically and efficiently regulate cell growth and death by the conditionally controlled antitoxin RelB switch, thereby serving as a novel counter-selectable cassette to establish the TCCRAS system. Using a single vector, such a system has been employed to perform in-frame deletion, functional knock-in, gene replacement, precise point mutation, large-scale insertion, and especially, deletion of the fragments up to 194.9 kb in B. subtilis. In addition, the biosynthesis of lycopene was first achieved in B. subtilis using TCCRAS to integrate a 5.4-kb fusion cluster (P_spac–crtI–crtE–crtB). The system was further adapted for gene knockdown and replacement, and large-scale deletion of the fragments up to 179.8 kb in C. glutamicum, with the mutation efficiencies increased by 0.8–1.0-fold compared to the conventional SacB method. TCCRAS thus holds promise as an effective and versatile genome-scale engineering technology for metabolic engineering and synthetic genomics research in a broad range of the Gram-positive bacteria.

中文翻译：

通过设计的毒素-抗毒素盒对细菌基因组进行编辑

以有效的方式操纵细菌基因组对于生物学和生物技术研究至关重要。在这里，我们将细菌TA系统重新编程为由抗毒素开关（TCCRAS）调控的毒素反向选择盒，用于在广泛研究和利用的革兰氏阳性细菌，枯草芽孢杆菌和谷氨酸棒杆菌中进行基因修饰。。在五个特征性的II型TA系统中，RelBE复合物可以通过有条件控制的抗毒素RelB开关特异性和有效地调节细胞的生长和死亡，从而用作建立TCCRAS系统的新型反向选择盒。使用单个载体，已将这种系统用于进行框内缺失，功能敲入，基因置换，精确点突变，大规模插入，尤其是缺失枯草芽孢杆菌中高达194.9 kb的片段。此外，番茄红素的生物合成首先在枯草芽孢杆菌中使用TCCRAS整合了一个5.4-kb的融合簇（P _spac – crtI – crtE – crtB）。）。该系统进一步适用于基因敲除和替换，以及在谷氨酸棒杆菌中大规模缺失高达179.8 kb的片段，与传统SacB方法相比，突变效率提高了0.8-1.0倍。因此，TCCRAS作为一种有效且用途广泛的基因组规模的工程技术，有望在广泛的革兰氏阳性细菌中用于代谢工程和合成基因组学研究。

更新日期：2017-01-17

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