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

• DOI: 10.1128/aem.02183-24
• Publication Date: 2025-03-26T13:14:38Z
• Authors: Jun-Zhe Zhang; Yu-Zhen Li; Zhi-Ning Xi; Yue Zhang; Zi-Yong Liu; Xiao-Qing Ma; Fu-Li Li
• Keywords: Metabolic Engineering; Genome Engineering; Gene knockout; CRISPR interference

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