Development of an in vivo methylation system for transformation of Ruminiclostridium cellulolyticum.

AIMS 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 Ruminiclostridium cellulolyticum avoiding its restriction system. METHODS AND RESULTS 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 2.6×103 ±0.23×103 CFU μg-1 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. CONCLUSIONS In this study, we generated an in vivo methylation system of R. cellulolyticum, allowing interspecies DNA transfer and improving transformation efficiency. SIGNIFICANCE AND IMPACT This research result will greatly facilitate the metabolic engineering of R. cellulolyticum for biofuel production directly from cellulose.