Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain， inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants， and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore， the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome， resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423， which has never been genetically engineered before. Firstly， the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept， our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid， which led to a sharp increase of transformation efficiencies.