Details

Autor(en) / Beteiligte

• Diallo, Mamou
• Hocq, Rémi
• Collas, Florent
• Chartier, Gwladys
• Wasels, François
• Wijaya, Hani Surya
• Werten, Marc W.T.
• Wolbert, Emil J.H.
• Kengen, Servé W.M.
• van der Oost, John
• Ferreira, Nicolas Lopes
• López-Contreras, Ana M.

Titel

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

Ist Teil von

• Methods (San Diego, Calif.), 2020-02, Vol.172, p.51-60

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2020

Quelle

MEDLINE

Beschreibungen/Notizen

• •A two-plasmid inducible CRISPR-Cas9 tool was fitted to Clostridium beijerinckii.•Deletion and insertion of genes in C. beijerinckii NCIMB 8052 were performed.•Gene deletion and plasmid curation in C. beijerinckii DSM 6423 were performed.•Asporogenous and cellulose-degrading C. beijerinckii mutants were obtained.•Plasmid curation in the DSM 6423 strain increased transformation efficiency. 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.