Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii
Abstract Background Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene tr...
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| Format: | Article |
| Language: | English |
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BMC
2019-05-01
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| Series: | Biotechnology for Biofuels |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13068-019-1448-1 |
| _version_ | 1818215834913341440 |
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| author | Gabriele Philipps Sebastian de Vries Stefan Jennewein |
| author_facet | Gabriele Philipps Sebastian de Vries Stefan Jennewein |
| author_sort | Gabriele Philipps |
| collection | DOAJ |
| description | Abstract Background Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways. Results We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source. Conclusions The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well. |
| first_indexed | 2024-12-12T06:42:23Z |
| format | Article |
| id | doaj.art-b841a20f6fa84812891742b909ff075e |
| institution | Directory Open Access Journal |
| issn | 1754-6834 |
| language | English |
| last_indexed | 2024-12-12T06:42:23Z |
| publishDate | 2019-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels |
| spelling | doaj.art-b841a20f6fa84812891742b909ff075e2022-12-22T00:34:18ZengBMCBiotechnology for Biofuels1754-68342019-05-0112111410.1186/s13068-019-1448-1Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahliiGabriele Philipps0Sebastian de Vries1Stefan Jennewein2Department for Industrial Biotechnology, Fraunhofer IME, Fraunhofer Institute for Molecular Biology and Applied Ecology IMEDepartment for Industrial Biotechnology, Fraunhofer IME, Fraunhofer Institute for Molecular Biology and Applied Ecology IMEDepartment for Industrial Biotechnology, Fraunhofer IME, Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAbstract Background Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways. Results We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source. Conclusions The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.http://link.springer.com/article/10.1186/s13068-019-1448-1Synthetic biologyMetabolic engineeringAcetogenConjugationInducible promoterHimar1 transposase |
| spellingShingle | Gabriele Philipps Sebastian de Vries Stefan Jennewein Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii Biotechnology for Biofuels Synthetic biology Metabolic engineering Acetogen Conjugation Inducible promoter Himar1 transposase |
| title | Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii |
| title_full | Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii |
| title_fullStr | Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii |
| title_full_unstemmed | Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii |
| title_short | Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii |
| title_sort | development of a metabolic pathway transfer and genomic integration system for the syngas fermenting bacterium clostridium ljungdahlii |
| topic | Synthetic biology Metabolic engineering Acetogen Conjugation Inducible promoter Himar1 transposase |
| url | http://link.springer.com/article/10.1186/s13068-019-1448-1 |
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