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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(2)) into multicarbon biochemicals. Genotype–phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic perf...

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Autores principales: Shin, Jongoh, Bae, Jiyun, Lee, Hyeonsik, Kang, Seulgi, Jin, Sangrak, Song, Yoseb, Cho, Suhyung, Cho, Byung-Kwan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9963998/
https://www.ncbi.nlm.nih.gov/pubmed/36716373
http://dx.doi.org/10.1073/pnas.2216244120
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author Shin, Jongoh
Bae, Jiyun
Lee, Hyeonsik
Kang, Seulgi
Jin, Sangrak
Song, Yoseb
Cho, Suhyung
Cho, Byung-Kwan
author_facet Shin, Jongoh
Bae, Jiyun
Lee, Hyeonsik
Kang, Seulgi
Jin, Sangrak
Song, Yoseb
Cho, Suhyung
Cho, Byung-Kwan
author_sort Shin, Jongoh
collection PubMed
description 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.
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spelling pubmed-99639982023-02-26 Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum Shin, Jongoh Bae, Jiyun Lee, Hyeonsik Kang, Seulgi Jin, Sangrak Song, Yoseb Cho, Suhyung Cho, Byung-Kwan Proc Natl Acad Sci U S A Biological Sciences 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. National Academy of Sciences 2023-01-30 2023-02-07 /pmc/articles/PMC9963998/ /pubmed/36716373 http://dx.doi.org/10.1073/pnas.2216244120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Shin, Jongoh
Bae, Jiyun
Lee, Hyeonsik
Kang, Seulgi
Jin, Sangrak
Song, Yoseb
Cho, Suhyung
Cho, Byung-Kwan
Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum
title Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum
title_full Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum
title_fullStr Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum
title_full_unstemmed Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum
title_short Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum
title_sort genome-wide crispri screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium eubacterium limosum
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9963998/
https://www.ncbi.nlm.nih.gov/pubmed/36716373
http://dx.doi.org/10.1073/pnas.2216244120
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