Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi Province, China.
Institute of Applied Chemistry, Shanxi University, Taiyuan, Shanxi Province, China.
J Appl Microbiol. 2022 Mar;132(3):1926-1935. doi: 10.1111/jam.15367. Epub 2021 Nov 24.
Ruminiclostridium cellulolyticum, an anaerobic cellulolytic bacterium producing an efficient cellulolytic extracellular complex named cellulosome, is a promising host for biofuel production from lignocellulose. This study aims to develop a rapid transformation method for R. cellulolyticum avoiding its restriction system.
The CceI restriction system is a major barrier to introduction of foreign DNA into R. cellulolyticum cells. To improve the transformation efficiency of R. cellulolyticum, the gene encoding CceI methyltransferase (M.CceI) of R. cellulolyticum H10 was functionally expressed in Escherichia coli, resulting in an in vivo methylation system for transformation of R. cellulolyticum. The electrotransformation experiments of R. cellulolyticum H10 with the E. coli-Clostridium shuttle plasmid pMTC6 showed that the transformation efficiency reached up to 2.6 × 10 ±0.23 × 10 CFU per μg plasmid DNA. The results demonstrated that the system is able to confer the M.CceI-specific DNA methylation pattern to its resident plasmid, which makes the plasmid resistant to the CceI restriction and efficiently transferred into R. cellulolyticum.
In this study, we generated an in vivo methylation system of R. cellulolyticum, allowing interspecies DNA transfer and improving transformation efficiency.
This research result will greatly facilitate the metabolic engineering of R. cellulolyticum for biofuel production directly from cellulose.
产高效纤维素酶细胞外复合酶系(cellulosome)的厌氧纤维素分解菌 Ruminiclostridium cellulolyticum 是木质纤维素生产生物燃料的有前途的宿主。本研究旨在开发一种快速转化方法，避免其限制系统。
CceI 限制系统是将外源 DNA 引入 R. cellulolyticum 细胞的主要障碍。为了提高 R. cellulolyticum 的转化效率，在大肠杆菌中功能性表达了 R. cellulolyticum H10 的 CceI 甲基转移酶（M.CceI）基因，构建了 R. cellulolyticum 的体内甲基化转化系统。用 E. coli-Clostridium 穿梭质粒 pMTC6 对 R. cellulolyticum H10 的电转化实验表明，转化效率高达 2.6±0.23×10 个 CFU/μg 质粒 DNA。结果表明，该系统能够赋予其驻留质粒 M.CceI 特异性 DNA 甲基化模式，使质粒对 CceI 限制具有抗性，并有效地转移到 R. cellulolyticum 中。
本研究构建了 R. cellulolyticum 的体内甲基化系统，实现了种间 DNA 转移，提高了转化效率。
该研究结果将极大地促进 R. cellulolyticum 代谢工程的发展，使其能够直接从纤维素生产生物燃料。
