Jongoh Shin ¹, Jiyun Bae ¹, Hyeonsik Lee ¹, Seulgi Kang ¹, Sangrak Jin ¹, Yoseb Song ¹, Suhyung Cho ^1,2, Byung-Kwan Cho ^1,2

¹Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.
²KAIST Institutes for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.

J.S. and J.B. contributed equally to this work.
To whom correspondence may be addressed : Byung-Kwan Cho

Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO₂) 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 CO₂-H₂ conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.