Targeted Deletions Uncover Previously Uncharacterised C 1 -fixing Cluster Genes as Essential for Autotrophy in Clostridium autoethanogenum

Recycling carbon oxides from industrial off-gases and gasified organic waste using gas fermentation offers a sustainable route for biomanufacturing of fuels and chemicals. Acetogens are attractive biocatalysts here and fix CO and CO 2 via the Wood-Ljungdahl pathway (WLP). However, the incomplete functional annotation of the associated C 1 -fixing gene cluster limits rational engineering of acetogen cell factories. Here, we investigated five highly conserved but previously uncharacterised C 1 -fixing cluster genes to quantify their contributions to the growth of the model-acetogen Clostridium autoethanogenum using targeted CRISPR/Cas9-mediated single-gene deletions, physiological characterisation, comparative computational structural analyses, CRISPRi, and functional genomics. Importantly, we identified acsV , lpdA , cooC , and cooC2 as essential for autotrophy but not heterotrophy, whereas deletion of gcvH reduced autotrophic growth and shifted carbon flux from acetate towards 2,3-butanediol. Amino acid supplementation experiments and computational analyses indicate that lpdA and gcvH do not primarily function in glycine or serine biosynthesis but instead fulfil specialised roles in WLP-associated C 1 and redox metabolism. Furthermore, genetic and physiological analyses showed that the two CooC-family ATPases, CooC and CooC2, have non-redundant roles in growth, with only cooC2 deletion impairing heterotrophy. Acetate supplementation tests revealed that Δ acsV cells retained metabolic activity despite abolished autotrophy, and computational analyses suggest a likely role for acsV in ATP-dependent corrinoid activation. Surprisingly, CRISPRi-mediated repression of acsV was translationally buffered, resulting in altered carbon partitioning without impairing autotrophy. Together, our findings provide insights into the physiological importance of the C 1 -fixing cluster genes in acetogens and support their metabolic engineering as gas-fermenting cell factories.