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

Clostridium ljungdahlii, as a model acetogen strain, represents a novel platform for biotechnological production for CO₂ 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.