Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering
Abstract Background The genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids,...
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| Format: | Article |
| Language: | English |
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BMC
2024-01-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-024-02301-8 |
| _version_ | 1797349585666965504 |
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| author | Maximilian Flaiz Anja Poehlein Wiebke Wilhelm Alexander Mook Rolf Daniel Peter Dürre Frank R. Bengelsdorf |
| author_facet | Maximilian Flaiz Anja Poehlein Wiebke Wilhelm Alexander Mook Rolf Daniel Peter Dürre Frank R. Bengelsdorf |
| author_sort | Maximilian Flaiz |
| collection | DOAJ |
| description | Abstract Background The genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids, but also due to their ability to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible, which makes it an interesting candidate to be an industrial biocatalyst. Results In this study, we examined genomic, phylogenetic, and physiologic features of E. limosum and the closest related species E. callanderi as well as E. maltosivorans. We sequenced the genomes of the six Eubacterium strains ‘FD’ (DSM 3662T), ‘Marburg’ (DSM 3468), ‘2A’ (DSM 2593), ‘11A’ (DSM 2594), ‘G14’ (DSM 107592), and ‘32’ (DSM 20517) and subsequently compared these with previously available genomes of the E. limosum type strain (DSM 20543T) as well as the strains ‘B2’, ‘KIST612’, ‘YI’ (DSM 105863T), and ‘SA11’. This comparison revealed a close relationship between all eleven Eubacterium strains, forming three distinct clades: E. limosum, E. callanderi, and E. maltosivorans. Moreover, we identified the gene clusters responsible for methanol utilization as well as genes mediating chain elongation in all analyzed strains. Subsequent growth experiments revealed that strains of all three clades can convert methanol and produce acetate, butyrate, and hexanoate via reverse β-oxidation. Additionally, we used a harmonized electroporation protocol and successfully transformed eight of these Eubacterium strains to enable recombinant plasmid-based expression of the gene encoding the fluorescence-activating and absorption shifting tag (FAST). Engineered Eubacterium strains were verified regarding their FAST-mediated fluorescence at a single-cell level using a flow cytometry approach. Eventually, strains ‘FD’ (DSM 3662T), ‘2A’ (DSM 2593), ‘11A’ (DSM 2594), and ‘32’ (DSM 20517) were genetically engineered for the first time. Conclusion Strains of E. limosum, E. callanderi, and E. maltosivorans are outstanding candidates as biocatalysts for anaerobic C1-substrate conversion into valuable biocommodities. A large variety of strains is genetically accessible using a harmonized electroporation protocol, and FAST can serve as a reliable fluorescent reporter protein to characterize genetically engineered cells. In total eleven strains have been assigned to distinct clades, providing a clear and updated classification. Thus, the description of respective Eubacterium species has been emended, improved, aligned, and is requested to be implemented in respective databases. |
| first_indexed | 2024-03-08T12:32:28Z |
| format | Article |
| id | doaj.art-90bfbf99a67f45b3a6b99ac2af856b4b |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-08T12:32:28Z |
| publishDate | 2024-01-01 |
| publisher | BMC |
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| series | Microbial Cell Factories |
| spelling | doaj.art-90bfbf99a67f45b3a6b99ac2af856b4b2024-01-21T12:40:26ZengBMCMicrobial Cell Factories1475-28592024-01-0123111910.1186/s12934-024-02301-8Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineeringMaximilian Flaiz0Anja Poehlein1Wiebke Wilhelm2Alexander Mook3Rolf Daniel4Peter Dürre5Frank R. Bengelsdorf6Laboratory of Microbiology, Wageningen University and ResearchGenomic and Applied Microbiology & Göttingen Genomics Laboratory, Georg-August UniversityInstitute of Microbiology and Biotechnology, University of UlmInstitute of Molecular Biology and Biotechnology of Prokaryotes, University of UlmGenomic and Applied Microbiology & Göttingen Genomics Laboratory, Georg-August UniversityInstitute of Microbiology and Biotechnology, University of UlmInstitute of Molecular Biology and Biotechnology of Prokaryotes, University of UlmAbstract Background The genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids, but also due to their ability to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible, which makes it an interesting candidate to be an industrial biocatalyst. Results In this study, we examined genomic, phylogenetic, and physiologic features of E. limosum and the closest related species E. callanderi as well as E. maltosivorans. We sequenced the genomes of the six Eubacterium strains ‘FD’ (DSM 3662T), ‘Marburg’ (DSM 3468), ‘2A’ (DSM 2593), ‘11A’ (DSM 2594), ‘G14’ (DSM 107592), and ‘32’ (DSM 20517) and subsequently compared these with previously available genomes of the E. limosum type strain (DSM 20543T) as well as the strains ‘B2’, ‘KIST612’, ‘YI’ (DSM 105863T), and ‘SA11’. This comparison revealed a close relationship between all eleven Eubacterium strains, forming three distinct clades: E. limosum, E. callanderi, and E. maltosivorans. Moreover, we identified the gene clusters responsible for methanol utilization as well as genes mediating chain elongation in all analyzed strains. Subsequent growth experiments revealed that strains of all three clades can convert methanol and produce acetate, butyrate, and hexanoate via reverse β-oxidation. Additionally, we used a harmonized electroporation protocol and successfully transformed eight of these Eubacterium strains to enable recombinant plasmid-based expression of the gene encoding the fluorescence-activating and absorption shifting tag (FAST). Engineered Eubacterium strains were verified regarding their FAST-mediated fluorescence at a single-cell level using a flow cytometry approach. Eventually, strains ‘FD’ (DSM 3662T), ‘2A’ (DSM 2593), ‘11A’ (DSM 2594), and ‘32’ (DSM 20517) were genetically engineered for the first time. Conclusion Strains of E. limosum, E. callanderi, and E. maltosivorans are outstanding candidates as biocatalysts for anaerobic C1-substrate conversion into valuable biocommodities. A large variety of strains is genetically accessible using a harmonized electroporation protocol, and FAST can serve as a reliable fluorescent reporter protein to characterize genetically engineered cells. In total eleven strains have been assigned to distinct clades, providing a clear and updated classification. Thus, the description of respective Eubacterium species has been emended, improved, aligned, and is requested to be implemented in respective databases.https://doi.org/10.1186/s12934-024-02301-8AcetogensAnaerobescallanderiEubacteriumFASTFluorescence |
| spellingShingle | Maximilian Flaiz Anja Poehlein Wiebke Wilhelm Alexander Mook Rolf Daniel Peter Dürre Frank R. Bengelsdorf Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering Microbial Cell Factories Acetogens Anaerobes callanderi Eubacterium FAST Fluorescence |
| title | Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering |
| title_full | Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering |
| title_fullStr | Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering |
| title_full_unstemmed | Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering |
| title_short | Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering |
| title_sort | refining and illuminating acetogenic eubacterium strains for reclassification and metabolic engineering |
| topic | Acetogens Anaerobes callanderi Eubacterium FAST Fluorescence |
| url | https://doi.org/10.1186/s12934-024-02301-8 |
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