Development of an in vivo methylation system for the solventogen Clostridium saccharobutylicum NCP 262 and analysis of two endonuclease mutants

•Using in vivo methylation of donor DNA, a triparental mating system for Clostridium saccharobutylicum was developed.•Mutants of the important solvent producer C. saccharobutylicum were constructed for the first time.•Knock-out mutants of the restriction systems of C. saccharobutylicum were constructed.•The influence of the individual restriction systems of C. saccharobutylicum on conjugation efficiency was studied. The genome of the biotechnologically important solventogenic Clostridium saccharobutylicum NCP 262 contains two operons coding for genes of presumed type I RM systems belonging to the families A and C. They represent a limiting factor for the development of transformation and conjugation protocols. We established an efficient triparental mating system to transfer DNA to C. saccharobutylicum by conjugation, which includes an in vivo methylation of the donor DNA. Furthermore we describe increased rates of conjugation in knock-out mutants of the restrictase subunits of both RM systems.