期刊
ACS SYNTHETIC BIOLOGY
卷 7, 期 3, 页码 822-831出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00287
关键词
toxin-antitoxin (TA) system; genome editing; large-scale deletion; Bacillus subtilis; Corynebacterium glutamicum
资金
- National Natural Science Foundation of China [31570083, 31170103]
- National Hi-Tech Research & Development Program of China [2014AA021203]
- Science and Technology Service Network Initiative [KFJ-EW-STS-078]
- Chinese Academy of Sciences [KGZD-EW-606]
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 ReIBE complex can specifically and efficiently regulate cell growth and death by the conditionally controlled antitoxin ReIB 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-spc-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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据