| Targeted gene engineering in Clostridium cellulolyticum H10 without methylation | |
| Cui, Gu-zhen1; Hong, Wei1; Zhang, Jie1; Li, Wen-li2; Feng, Yingang1; Liu, Ya-jun1; Cui, Qiu1 | |
| 2012-06-01 | |
| 发表期刊 | JOURNAL OF MICROBIOLOGICAL METHODS
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| 卷号 | 89期号:3页码:201-208 |
| 摘要 | Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative MspI endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C. cellulolyticum mutant H10ΔmspI lost the MspI endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10ΔmspI without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10ΔmspI, double mutants H10ΔmspIΔldh and H10ΔmspIΔack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10ΔmspI, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10ΔmspI constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level.; Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative Mspl endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C cellulolyticum mutant H10 Delta mspl lost the Mspl endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10 Delta mspl without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10 Delta mspl, double mutants H10 Delta mspl Delta ldh and H10 Delta mspl Delta ack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10 Delta mspl, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10 Delta mspl constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level. (c) 2012 Elsevier B.V. All rights reserved. |
| 文章类型 | Article |
| 关键词 | Methylation-free Clostron Mspl Oxygen-independent Green Fluorescence Protein Lactate Dehydrogenase Acetate Kinase |
| 学科领域 | 代谢物组学 |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| DOI | 10.1016/j.mimet.2012.02.015 |
| 关键词[WOS] | GROUP-II INTRONS ; ESCHERICHIA-COLI ; BIOFUEL PRODUCTION ; ACETOBUTYLICUM ; TRANSFORMATION ; RESTRICTION ; DNA ; PROTEIN ; THERMOCELLUM ; EXPRESSION |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Biochemistry & Molecular Biology ; Microbiology |
| WOS类目 | Biochemical Research Methods ; Microbiology |
| WOS记录号 | WOS:000304506700009 |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qibebt.ac.cn/handle/337004/1225 |
| 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, Key Lab Biofuels, Qingdao 266101, Shandong, Peoples R China 2.Ocean Univ China, Key Lab Marine Drugs, Minist Educ, Sch Med & Pharm, Qingdao, Shandong, Peoples R China |
| 推荐引用方式 GB/T 7714 | Cui, Gu-zhen,Hong, Wei,Zhang, Jie,et al. Targeted gene engineering in Clostridium cellulolyticum H10 without methylation[J]. JOURNAL OF MICROBIOLOGICAL METHODS,2012,89(3):201-208. |
| APA | Cui, Gu-zhen.,Hong, Wei.,Zhang, Jie.,Li, Wen-li.,Feng, Yingang.,...&Cui, Qiu.(2012).Targeted gene engineering in Clostridium cellulolyticum H10 without methylation.JOURNAL OF MICROBIOLOGICAL METHODS,89(3),201-208. |
| MLA | Cui, Gu-zhen,et al."Targeted gene engineering in Clostridium cellulolyticum H10 without methylation".JOURNAL OF MICROBIOLOGICAL METHODS 89.3(2012):201-208. |
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