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Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C(1)) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO(2)) in...

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Autores principales: Nwaokorie, Ugochi Jennifer, Reinmets, Kristina, de Lima, Lorena Azevedo, Pawar, Pratik Rajendra, Shaikh, Kurshedaktar Majibullah, Harris, Audrey, Köpke, Michael, Valgepea, Kaspar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10230548/
https://www.ncbi.nlm.nih.gov/pubmed/37265994
http://dx.doi.org/10.3389/fbioe.2023.1167892
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author Nwaokorie, Ugochi Jennifer
Reinmets, Kristina
de Lima, Lorena Azevedo
Pawar, Pratik Rajendra
Shaikh, Kurshedaktar Majibullah
Harris, Audrey
Köpke, Michael
Valgepea, Kaspar
author_facet Nwaokorie, Ugochi Jennifer
Reinmets, Kristina
de Lima, Lorena Azevedo
Pawar, Pratik Rajendra
Shaikh, Kurshedaktar Majibullah
Harris, Audrey
Köpke, Michael
Valgepea, Kaspar
author_sort Nwaokorie, Ugochi Jennifer
collection PubMed
description Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C(1)) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO(2)) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C(1)-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions—hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950)—directly adjacent and expressed at similar levels to the C(1)-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆hp and ∆nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO(2) and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C(1)-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.
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spelling pubmed-102305482023-06-01 Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum Nwaokorie, Ugochi Jennifer Reinmets, Kristina de Lima, Lorena Azevedo Pawar, Pratik Rajendra Shaikh, Kurshedaktar Majibullah Harris, Audrey Köpke, Michael Valgepea, Kaspar Front Bioeng Biotechnol Bioengineering and Biotechnology Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C(1)) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO(2)) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C(1)-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions—hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950)—directly adjacent and expressed at similar levels to the C(1)-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆hp and ∆nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO(2) and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C(1)-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation. Frontiers Media S.A. 2023-05-15 /pmc/articles/PMC10230548/ /pubmed/37265994 http://dx.doi.org/10.3389/fbioe.2023.1167892 Text en Copyright © 2023 Nwaokorie, Reinmets, de Lima, Pawar, Shaikh, Harris, Köpke and Valgepea. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Nwaokorie, Ugochi Jennifer
Reinmets, Kristina
de Lima, Lorena Azevedo
Pawar, Pratik Rajendra
Shaikh, Kurshedaktar Majibullah
Harris, Audrey
Köpke, Michael
Valgepea, Kaspar
Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum
title Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum
title_full Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum
title_fullStr Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum
title_full_unstemmed Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum
title_short Deletion of genes linked to the C(1)-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum
title_sort deletion of genes linked to the c(1)-fixing gene cluster affects growth, by-products, and proteome of clostridium autoethanogenum
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10230548/
https://www.ncbi.nlm.nih.gov/pubmed/37265994
http://dx.doi.org/10.3389/fbioe.2023.1167892
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