Targeted Deletions Uncover Previously Uncharacterised C1-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 CO2 via the Wood-Ljungdahl pathway (WLP). However, the incomplete functional annotation of the associated C1-fixing gene cluster limits rational engineering of acetogen cell factories. Here, we investigated five highly conserved but previously uncharacterised C1-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 C1 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 {Delta}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 C1-fixing cluster genes in acetogens and support their metabolic engineering as gas-fermenting cell factories.