Inducible promoters of bacterial microcompartments improve the CRISPR/Cas9 tools for efficient metabolic engineering of Clostridium ljungdahlii

ABSTRACT Clostridium ljungdahlii , as a model acetogen strain, represents a novel platform for biotechnological production for CO 2 fixation. The genome of C. ljungdahlii harbors two gene loci associated with glycyl radical enzyme-associated microcompartments (GRMs), which are predicted to play essential roles in choline and 1,2-propanediol (1,2-PD) metabolism. This study validated the functions of these GRM loci and identified two inducible promoters, of which P choline1 was induced by choline, while P 1,2-PD was induced by 1,2-PD. Subsequently, the highly expressed P 1,2-PD and tightly controlled P choline1 were applied to improve CRISPR/Cas9 gene editing tools. Specifically, P 1,2-PD was used to develop a highly efficient gene knockout tool based on an all-in-one plasmid, achieving 100% deletion efficiency for multiple genes, including pyrE , pduS , aor2 , and eutT . On the other hand, the cas9 gene was integrated downstream of P choline1 into the genome. The integrated cas9 efficiently mediated gene editing in C. ljungdahlii by introducing plasmids containing a gRNA cassette along with the relevant homology arms. This was exemplified by the construction of the Δbdh::pdc strain, where the 2,3-butanediol dehydrogenase gene was replaced with a pyruvate decarboxylase gene from Zymomonas mobilis and the 3-HB Syn KI strain, in which an artificial 3-hydroxybutyric acid synthesis pathway was inserted into the genome. This study highlights the effectiveness and convenience of the inducible CRISPR/Cas9 gene editing systems, thereby enriching the CRISPR/Cas toolkit in acetogens. IMPORTANCE A CRISPR/Cas9 genetic tool controlled by a constitutive promoter has been developed for precise gene deletion in Clostridium ljungdahlii . However, its efficiency was hindered by the toxicity resulting from the constitutive expression of cas9 and the large plasmids, leading to a low overall success rate. Inducible promoters, which allow for the transcription of target genes to be switched on and off in the presence or absence of inducers, have a broad range of applications. In this study, we identify two inducible promoters and apply them to enhance the CRISPR/Cas9 tools. The improved CRISPR/Cas9 tools facilitate gene editing with high efficiency, potentially playing significant roles in advancing genetic research and metabolic engineering of C. ljungdahlii .