A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.

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.

The B12-Radical SAM Enzyme PoyC Catalyzes Valine Cß-Methylation during Polytheonamide Biosynthesis.

Genomic and metagenomic investigations have recently led to the delineation of a novel class of natural products called ribosomally synthesized and post-translationally modified peptides (RiPPs). RiPPs are ubiquitous among living organisms and include pharmaceutically relevant compounds such as antibiotics and toxins. A prominent example is polytheonamide A, which exhibits numerous post-translational modifications, some of which were unknown in ribosomal peptides until recently. Among these post-translational modifications, C-methylations have been proposed to be catalyzed by two putative radical S-adenosylmethionine (rSAM) enzymes, PoyB and PoyC. Here we report the in vitro activity of PoyC, the first B12-dependent rSAM enzyme catalyzing peptide Cß-methylation. We show that PoyC catalyzes the formation of S-adenosylhomocysteine and 5'-deoxyadenosine and the transfer of a methyl group to l-valine residue. In addition, we demonstrate for the first time that B12-rSAM enzymes have a tightly bound MeCbl cofactor that during catalysis transfers a methyl group originating from S-adenosyl-l-methionine. Collectively, our results shed new light on polytheonamide biosynthesis and the large and emerging family of B12-rSAM enzymes.