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Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system
Cui, Gu-Zhen1; Zhang, Jie1,3; Hong, Wei1,3; Xu, Chenggang1; Feng, Yingang1; Cui, Qiu1,2; Liu, Ya-Jun1
2014
发表期刊APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷号98期号:1页码:313-323
摘要

Clostridium includes a number of species, such as thermophilic Clostridium thermocellum and mesophilic Clostridium cellulolyticum, producing biofuels and chemicals from lignocellulose, while genetic engineering is necessary to improve wild-type strains to fulfill the requirement of industrialization. ClosTron system is widely used in the gene targeting of Clostridium because of its high efficiency and operability. However, the targetron plasmid present in cell hinders the successive gene disruption. To solve this problem, a pyrF-based screening system was developed and implemented in C. cellulolyticum strain H10 in this study for efficient targetron plasmid curing. The screening system was composed of a pyrF-deleted cell chassis (H10 Delta pyrF) constructed via homologous recombination and a PyrF expression cassette located in a targetron plasmid containing an erythromycin resistance gene. With the screening system, the gene targeting could be achieved following a two-step procedure, including the first step of gene disruption through targetron transformation and erythromycin selection and the second step of plasmid curing by screening with 5-fluoroorotic acid. To test the developed screening system, successive inactivation of the major cellulosomal exocellulase Cel48F and the scaffoldin protein CipC was achieved in C. cellulolyticum, and the efficient plasmid curing was confirmed. With the assistance of the pyrF-based screening system, the targetron plasmid-cured colonies can be rapidly selected by one-plate screening instead of traditional days' unguaranteed screening, and the successive gene disruption becomes accomplishable with ClosTron system with improved stability and efficiency, which may promote the metabolic engineering of Clostridium species aiming at enhanced production of biofuels and chemicals.

文章类型Article
关键词Clostridium Cellulolyticum Clostron Targetron Plasmid Curing Pyrf
WOS标题词Science & Technology ; Life Sciences & Biomedicine
DOI10.1007/s00253-013-5330-y
关键词[WOS]GROUP-II INTRONS ; BACTERIA ; DNA ; TRANSFORMATION ; MANIPULATION ; CELLULOSE ; SELECTION ; STRAIN ; THERMOCELLUM ; RESTRICTION
收录类别SCI
语种英语
WOS研究方向Biotechnology & Applied Microbiology
WOS类目Biotechnology & Applied Microbiology
WOS记录号WOS:000329093600028
引用统计
被引频次:21[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.qibebt.ac.cn/handle/337004/6119
专题代谢物组学研究组
作者单位1.Chinese Acad Sci, Qingdao Inst BioEnergy & Bioproc Technol, Shandong Prov Key Lab Energy Genet, Qingdao 266101, Shandong, Peoples R China
2.Chinese Acad Sci, Qingdao Inst Bioenergy & Bioproc Technol, Key Lab Biofuels, Qingdao 266101, Shandong, Peoples R China
3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
推荐引用方式
GB/T 7714
Cui, Gu-Zhen,Zhang, Jie,Hong, Wei,et al. Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system[J]. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY,2014,98(1):313-323.
APA Cui, Gu-Zhen.,Zhang, Jie.,Hong, Wei.,Xu, Chenggang.,Feng, Yingang.,...&Liu, Ya-Jun.(2014).Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system.APPLIED MICROBIOLOGY AND BIOTECHNOLOGY,98(1),313-323.
MLA Cui, Gu-Zhen,et al."Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system".APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 98.1(2014):313-323.
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