Pooled CRISPR interference screening identifies crucial transcription factors in gas-fermentingClostridium ljungdahlii

Abstract

Gas-fermentingClostridiumspecies hold tremendous promise for one-carbon biomanufacturing. To unlock their full potential, it is crucial to unravel and optimize the intricate regulatory networks that govern these organisms; however, this aspect is currently underexplored. In this study, we employed pooled CRISPR interference (CRISPRi) screening to uncover a wide range of functional transcription factors (TFs) inClostridium ljungdahlii, a representative species of gas-fermentingClostridium, with a special focus on the TFs associated with the utilization of carbon resources. Among the 425 TF candidates, we identified 75 and 68 TF genes affecting the heterotrophic and autotrophic growth ofC. ljungdahlii, respectively. We directed our attention on two of the screened TFs, NrdR and DeoR, and revealed their pivotal roles in the regulation of deoxyribonucleotides (dNTPs) supply, carbon fixation, and product synthesis inC. ljungdahlii, thereby influencing the strain performance in gas fermentation. Based on this, we proceeded to optimize the expression ofdeoRinC. ljungdahliiby adjusting its promoter strength, leading to improved growth rate and ethanol synthesis ofC. ljungdahliiwhen utilizing syngas. This study highlights the effectiveness of pooled CRISPRi screening in gas-fermentingClostridiumspecies, expanding the horizons for functional genomic research in these industrially important bacteria.