Targeted gene engineering in Clostridium cellulolyticum H10 without methylation

• Published in: Journal of microbiological methods Vol. 89; no. 3; pp. 201 - 208
• Main Authors: Cui, Gu-zhen, Hong, Wei, Zhang, Jie, Li, Wen-li, Feng, Yingang, Liu, Ya-jun, Cui, Qiu
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.06.2012; Elsevier
• Subjects: Acetate kinase; biofuels; Biological and medical sciences; cellulose; Clostridium cellulolyticum; Clostridium cellulolyticum - genetics; ClosTron; Deoxyribonuclease HpaII - genetics; Deoxyribonuclease HpaII - metabolism; DNA; DNA Methylation; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; fluorescence; Fundamental and applied biological sciences. Psychology; Gene Knockout Techniques; Gene Targeting; genes; Genes, Reporter; genetic engineering; Genetic Engineering - methods; green fluorescent protein; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - genetics; heterologous gene expression; Lactate dehydrogenase; metabolic engineering; Methylation-free; Microbiology; Molecular Sequence Data; MspI; mutants; mutation; Oxygen-independent green fluorescence protein; Sequence Analysis, DNA; Transformation, Bacterial; Oxygen-independent green fluorescence protein; Lactate dehydrogenase; Methylation-free; Acetate kinase; MspI; ClosTron; Oxygen; Oxygen-independent green fluorescence; Enzyme; Transferases; Mspl; Clostridiales; Clostridium cellulolyticum; Dehydrogenase; L-Lactate dehydrogenase; Protein; Gene; Kinase; Clostridiaceae; Bacteria; Oxidoreductases; Methylation
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2012.02.015

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. ► A methylation-free genetic platform was developed in Clostridium cellulolyticum H10. ► The platform has high transformation efficiency, screening rate and stability. ► The mutant H10ΔmspI lost MspI activity and accepted unmethylated DNA efficiently. ► H10ΔmspI is capable of heterologous gene expression and serial gene inactivation. ► An anaerobic green fluorescent reporter system was constructed in H10ΔmspI.