title: Bacterial Genome Editing via a Designed Toxin-Antitoxin Cassette.
abstract: 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 ( Pspac- 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.
authors.affiliation: 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.
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Deng
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Qinyun
authors.initials: Q
authors.lastname: Sun
authors.affiliation: 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.
authors.forename: Hua
authors.initials: H
authors.lastname: Bai
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Zhaopeng
authors.initials: Z
authors.lastname: Sun
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Xiuling
authors.initials: X
authors.lastname: Shang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liang
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.
authors.forename: Shuwen
authors.initials: S
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Toxicology & Medical Countermeasures , Beijing Institute of Pharmacology & Toxicology , Beijing 100850 , China.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Che
authors.affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 , China.|Savaid medical school , University of Chinese Academy of Sciences , Beijing 100049 , China.
authors.forename: Tingyi
authors.identifier: 0000-0003-2583-7088
authors.initials: T
authors.lastname: Wen
mesh_terms: D000992:Antitoxins / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D048230:Corynebacterium glutamicum / Q000235:genetics*; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D005798:Genes, Bacterial; D005818:Genetic Engineering; D016680:Genome, Bacterial; D009876:Operon / Q000235:genetics; D017354:Point Mutation / Q000235:genetics
chemical_list: D000992:Antitoxins; D001427:Bacterial Toxins
keywords: Bacillus subtilis; Corynebacterium glutamicum; genome editing; large-scale deletion; toxin−antitoxin (TA) system