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

Authors
  • Shin, Jongoh
  • Bae, Jiyun
  • Lee, Hyeonsik
  • Kang, Seulgi
  • Jin, Sangrak
  • Song, Yoseb
  • Cho, Suhyung
  • Cho, Byung-Kwan
Type
Published Article
Journal
Proceedings of the National Academy of Sciences
Publisher
Proceedings of the National Academy of Sciences
Publication Date
Jan 30, 2023
Volume
120
Issue
6
Identifiers
DOI: 10.1073/pnas.2216244120
PMID: 36716373
PMCID: PMC9963998
Source
PubMed Central
Keywords
Disciplines
  • Biological Sciences
  • Microbiology
License
Unknown

Abstract

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.

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