A CRISPR interference system for tunable gene expression integrated with a promoter library for <i>Eubacterium callanderi</i> KIST612, an acetogen of functional diversity and versatility

ABSTRACT Acetogens are key biocatalysts for carbon-neutral biorefineries, yet their metabolic engineering is limited by the lack of tunable transcriptional regulation systems. Here, we developed a synthetic promoter library for Eubacterium callanderi KIST612 and integrated it with a CRISPR interference (CRISPRi) system to establish precise and scalable gene regulation. Motif analysis of 3,109 putative native promoters revealed conserved and semi-conserved −35 and −10 elements, which were used to construct a promoter library spanning a &gt;20-fold dynamic range of transcriptional strengths. The system was validated by knockdown of pyrF , where promoter strength directly determined repression efficiency ( R 2 = 0.92), with high-strength promoters achieving near-complete gene silencing. Application to lactate dehydrogenase ( ldh ) revealed that increasing promoter strength progressively reduced lactate production from 93.3% to 0.0% of control. This study establishes a versatile synthetic promoter-CRISPRi platform tailored for acetogens, enabling precise control of gene expression and mechanistic dissection of redox metabolism. IMPORTANCE Transitioning to a carbon-neutral economy requires biocatalysts that can efficiently convert waste-derived substrates into valuable products. Acetogens are industrially relevant organisms for gas fermentation, but the lack of genetic toolkits tailored to their physiology has constrained metabolic engineering. We present the first synthetic promoter-CRISPRi platform specifically optimized for Eubacterium callanderi KIST612, a model acetogen with high industrial potential. This system provides tunable and predictable regulation of gene expression, extending from mild repression to a near-complete knockdown that could alternate gene deletion systems. This system could be used for not only advancing fundamental understanding of acetogen physiology but also providing a broadly applicable genetic toolbox for precision engineering of sustainable microbial biorefineries.