Genome-wide CRISPRi screening of Eubacterium limosum

Eubacterium limosum is one of the acetogenic bacteria, which important biocatalysts capable of C1 feedstock into value-added multi-carbon chemicals through gas-fermentation. To investigate genotype-phenotype relationships under heterotrophic and autotrophic conditions, we here report a genome-scale CRISPRi screen, where almost genes were targeted for transcriptional suppression. By investigating ?tness contributions of all genes, we could obtain a rich dataset revealing features of the gene essentiality under heterotrophic and autotrophic growth conditions. Our data reveal that the Wood-Ljungdahl pathway is essential in both heterotrophic and autotrophic growth and the unknown gene set that is important for autotrophic acetogenesis. Furthermore, we validated several mutants that increase the autotrophic growth rates up to 1.98-fold under autotrophic growth conditions. The results provide insights into accelerating the understanding of acetogenic metabolism and genome engineering in acetogens.