Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum

Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO ) into multicarbon biochemicals. Genotype-phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic perfor...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2023-02, Vol.120 (6), p.e2216244120-e2216244120
Main Authors: Shin, Jongoh, Bae, Jiyun, Lee, Hyeonsik, Kang, Seulgi, Jin, Sangrak, Song, Yoseb, Cho, Suhyung, Cho, Byung-Kwan
Format: Article
Language:English
Subjects:
Acetogenesis
Acetoin
Autotrophic Processes
Bacteria
Bioconversion
Biological Sciences
Biosynthesis
Carbon dioxide
Carbon Dioxide - metabolism
CRISPR
Eubacterium - genetics
Eubacterium - metabolism
Eurytium limosum
Fitness
Gene silencing
Genes
Genome, Bacterial
Genomes
Genotypes
Growth rate
Membrane proteins
Membranes
Metabolic engineering
Phenotypes
Protein biosynthesis
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Summary:Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO ) 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 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 . 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 -H conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2216244120