Transcriptional modulation of the global regulator CodY using a conditional CRISPRi system in Bacillus licheniformis

Bacillus licheniformis is an industrially significant microorganism known for its broad carbon source utilization compared to other bacteria. However, the mechanisms underlying this utilization are tightly controlled by global regulatory proteins such as CodY, and the details of these mechanisms remain elusive. This poses challenges for metabolic engineering efforts. In this study, we used the urease encoding gene as a reporter to establish a CRISPRi system based on conditional dCas9 expression in B. licheniformis . The induction with mannose resulted in an 84% transcriptional inhibition of ureA , and a 57% reduction in urease activity, confirming the system's successful construction. We designed three different sgRNA sites within the 5'-end coding region of the codY gene to achieve varying degrees of protein expression knockdown. The results showed that a 10–75% knockdown of codY led to a 23–87% decrease in the maximum specific uptake rates of glucose and maltose. Concurrently, the accumulation of carbon overflow metabolites such as 2,3-butanediol (2,3-BDO) and acetate decreased by 38% and 26%, respectively. These findings enhance our understanding of CodY’s regulatory role in catabolism and metabolism. The CRISPRi system with conditional dCas9 expression developed here serves as an effective synthetic biology tool for metabolic pathway engineering.