Deletion of genes linked to the C1-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 (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into v...
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Frontiers Media S.A.
2023-05-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1167892/full |
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| author | Ugochi Jennifer Nwaokorie Kristina Reinmets Lorena Azevedo de Lima Pratik Rajendra Pawar Kurshedaktar Majibullah Shaikh Audrey Harris Michael Köpke Kaspar Valgepea |
| author_facet | Ugochi Jennifer Nwaokorie Kristina Reinmets Lorena Azevedo de Lima Pratik Rajendra Pawar Kurshedaktar Majibullah Shaikh Audrey Harris Michael Köpke Kaspar Valgepea |
| author_sort | Ugochi Jennifer Nwaokorie |
| collection | DOAJ |
| description | Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-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 C1-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 CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-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. |
| format | Article |
| id | doaj-art-c8d03f33be564159bc2a137ea2ea806a |
| institution | OA Journals |
| issn | 2296-4185 |
| language | English |
| publishDate | 2023-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj-art-c8d03f33be564159bc2a137ea2ea806a2025-08-20T02:26:46ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-05-011110.3389/fbioe.2023.11678921167892Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenumUgochi Jennifer Nwaokorie0Kristina Reinmets1Lorena Azevedo de Lima2Pratik Rajendra Pawar3Kurshedaktar Majibullah Shaikh4Audrey Harris5Michael Köpke6Kaspar Valgepea7ERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, EstoniaERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, EstoniaERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, EstoniaERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, EstoniaERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, EstoniaLanzaTech Inc., Skokie, IL, United StatesLanzaTech Inc., Skokie, IL, United StatesERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, EstoniaGas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-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 C1-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 CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-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.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1167892/fullgas fermentationacetogenCRISPR/Cas9genetic engineeringmetabolomicsproteomics |
| spellingShingle | Ugochi Jennifer Nwaokorie Kristina Reinmets Lorena Azevedo de Lima Pratik Rajendra Pawar Kurshedaktar Majibullah Shaikh Audrey Harris Michael Köpke Kaspar Valgepea Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum Frontiers in Bioengineering and Biotechnology gas fermentation acetogen CRISPR/Cas9 genetic engineering metabolomics proteomics |
| title | Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum |
| title_full | Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum |
| title_fullStr | Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum |
| title_full_unstemmed | Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum |
| title_short | Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum |
| title_sort | deletion of genes linked to the c1 fixing gene cluster affects growth by products and proteome of clostridium autoethanogenum |
| topic | gas fermentation acetogen CRISPR/Cas9 genetic engineering metabolomics proteomics |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1167892/full |
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