C1 gases, including carbon dioxide (CO2) and carbon monoxide (CO), are major contributors to climate crisis. Numerous studies have been conducted to fix and recycle C1 gases in order to solve this problem. Among them, the use of microorganisms as biocatalysts to convert C1 gases to value-added chemicals is a promising solution. Acetogenic bacteria (acetogens) have received attention as high-potential biocatalysts owing to their conserved Wood–Ljungdahl (WL) pathway, which fixes not only CO2 but also CO. Although some metabolites have been produced via C1 gas fermentation on an industrial scale, the conversion of C1 gases to produce various biochemicals by engineering acetogens has been limited. The energy limitation of acetogens is one of the challenges to overcome, as their metabolism operates at a thermodynamic limit, and the low solubility of gaseous substrates results in a limited supply of cellular energy. This review provides strategies for developing efficient platform strains for C1 gas conversion, focusing on engineering the WL pathway. Supplying liquid C1 substrates, which can be obtained from CO2, or electricity is introduced as a strategy to overcome the energy limitation. Future prospective approaches on engineering acetogens based on systems and synthetic biology approaches are also discussed.

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Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO 2 ) into multicarbon biochemicals. Genotype–phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic performance; however, systemic investigation on the fitness contribution of individual gene has been limited. Here, we report genome-scale CRISPR interference screening using 41,939 guide RNAs designed from the E. limosum genome, one of the model acetogenic species, where all genes were targeted for transcriptional suppression. We investigated the fitness contributions of 96% of the total genes identified, revealing the gene fitness and essentiality for heterotrophic and autotrophic metabolisms. Our data show that the Wood–Ljungdahl pathway, membrane regeneration, membrane protein biosynthesis, and butyrate synthesis are essential for autotrophic acetogenesis in E. limosum . Furthermore, we discovered genes that are repression targets that unbiasedly increased autotrophic growth rates fourfold and acetoin production 1.5-fold compared to the wild-type strain under CO 2 -H 2 conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.