title: Targeted gene engineering in Clostridium cellulolyticum H10 without methylation.
abstract: 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.
authors.affiliation: Shandong Provincial Key Laboratory of Energy Genetics, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, PR China.
authors.forename: Gu-zhen
authors.initials: GZ
authors.lastname: Cui
authors.forename: Wei
authors.initials: W
authors.lastname: Hong
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Wen-li
authors.initials: WL
authors.lastname: Li
authors.forename: Yingang
authors.initials: Y
authors.lastname: Feng
authors.forename: Ya-jun
authors.initials: YJ
authors.lastname: Liu
authors.forename: Qiu
authors.initials: Q
authors.lastname: Cui
mesh_terms: D046973:Ruminiclostridium cellulolyticum / Q000235:genetics*; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019003:Deoxyribonuclease HpaII / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D049452:Green Fluorescent Proteins; D019003:Deoxyribonuclease HpaII