Abstract

The efficient utilization of industrial one-carbon (C1) gases, such as carbon dioxide (CO 2 ) and carbon monoxide (CO), has attracted widespread interest in recent years. Clostridium ljungdahlii , a key chemoautotrophic bacterium, can ferment syngas (a mixture of CO 2 , CO and H 2 ) into value-added chemicals, making it a subject of extensive research. However, despite advances in genetic tool development for C. ljungdahlii , the rapid identification of genes underlying crucial cellular functions remains a bottleneck, hindering both a deeper understanding and successful genetic engineering of this bacterium. In this study, we constructed a genome-wide random mutagenesis library of C. ljungdahlii using a mariner transposon system, enabling the identification of numerous functional genes associated with cellular fitness to syngas. We overcame the library's relatively low plasmid curing efficiency by employing an IPTG-regulated conditional replicon. Subsequent high-throughput transposon insertion sequencing (Tn-seq) of this library allowed for the rapid exploration of a broad set of genes potentially involved in growth on syngas. Experimental validation of these candidate genes revealed several that affect the production of ethanol and acetate (two major metabolites) of C. ljungdahlii in gas fermentation. The data generated in this study provide a valuable resource for future research and optimization of C. ljungdahlii . Furthermore, the strategy presented here can be applied to identify genes associated with other important phenotypes in this bacterium.