Interactive Regulation of Formate Dehydrogenase during CO₂ Fixation in Gas-Fermenting Bacteria

Protein lysine acetylation, a prevalent posttranslational modification, regulates numerous crucial biological processes in cells. Nevertheless, how lysine acetylation interacts with other types of regulation to coordinate metabolism remains largely unknown owing to the complexity of the process. Here, using a representative gas-fermenting bacterium, Clostridium ljungdahlii, we revealed a novel regulatory mechanism that employs both the lysine acetylation and transcriptional regulation systems to interactively control CO₂ fixation, a key biological process for utilizing this one-carbon gas. A dominant lysine acetyltransferase/deacetylase system, At2/Dat1, was identified and found to regulate FDH1 (formate dehydrogenase responsible for CO₂ fixation) activity via a crucial acetylation site (lysine-29). Notably, the global transcription factor CcpA was also shown to be regulated by At2/Dat1; in turn, CcpA could directly control At2 expression, thus indicating an unreported interaction mode between the acetylation system and transcription factors. Moreover, CcpA was observed to negatively regulate FDH1 expression, which, when combined with At2/Dat1, leads to the collaborative regulation of this enzyme. Based on this concept, we reconstructed the regulatory network related to FDH1, realizing significantly increased CO₂ utilization by C. ljungdahliiIMPORTANCE Microbial CO₂ fixation and conversion constitute a potential solution to both utilization of greenhouse gas or industrial waste gases and sustainable production of bulk chemicals and fuels. Autotrophic gas-fermenting bacteria play central roles in this bioprocess. This study provides new insights regarding the metabolic regulatory mechanisms underlying CO₂ reduction in Clostridium ljungdahlii, a representative gas-fermenting bacterium. A critical formate dehydrogenase (FDH1) responsible for fixing CO₂ and a dominant reversible lysine acetylation system, At2/Dat1, were identified. Furthermore, FDH1 was found to be interactively regulated by both the At2/Dat1 system and the global transcriptional factor CcpA, and the two regulatory systems are mutually restricted. Reconstruction of this multilevel metabolic regulatory module led to improved CO₂ metabolism by C. ljungdahlii These findings not only substantively expand our understanding but also provide a potentially useful metabolic engineering strategy for microbial carbon fixation.