A CRISPR interference system for tunable gene expression integrated with a promoter library for Eubacterium callanderi 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 >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.