Wu Jie, Deng Aihua, Sun Qinyun, Bai Hua, Sun Zhaopeng, Shang Xiuling, Zhang Yun, Liu Qian, Liang Yong, Liu Shuwen, Che Yongsheng, Wen Tingyi
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
University of Chinese Academy of Sciences , Beijing 100049 , China.
ACS Synth Biol. 2018 Mar 16;7(3):822-831. doi: 10.1021/acssynbio.6b00287. Epub 2017 Jan 26.
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 - 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.
以高效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。在此，我们将细菌TA系统重新编程为受抗毒素开关调控的毒素反向选择盒（TCCRAS），用于在广泛研究和应用的革兰氏阳性菌枯草芽孢杆菌和谷氨酸棒杆菌中进行基因改造。在五个已表征的II型TA系统中，RelBE复合物可通过条件控制的抗毒素RelB开关特异性且有效地调节细胞生长和死亡，从而作为一种新型反向选择盒来建立TCCRAS系统。使用单个载体，该系统已用于在枯草芽孢杆菌中进行框内缺失、功能性敲入、基因替换、精确点突变、大规模插入，尤其是长达194.9 kb片段的缺失。此外，首次在枯草芽孢杆菌中利用TCCRAS整合一个5.4 kb的融合簇（P - crtI - crtE - crtB）实现了番茄红素的生物合成。该系统进一步适用于基因敲低和替换，以及在谷氨酸棒杆菌中长达179.8 kb片段的大规模缺失，与传统的SacB方法相比，突变效率提高了0.8 - 1.0倍。因此，TCCRAS有望成为一种有效且通用的基因组规模工程技术，用于广泛的革兰氏阳性菌中的代谢工程和合成基因组学研究。
