CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313
Summary Hungateiclostridium thermocellum DSM 1313 has biotechnological potential as a whole‐cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been hampered due to the lack of simple and high‐throughput genome engineering too...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Wiley
2020-03-01
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| Series: | Microbial Biotechnology |
| Online Access: | https://doi.org/10.1111/1751-7915.13516 |
| _version_ | 1818042613533507584 |
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| author | Joyshree Ganguly Maria Martin‐Pascual Richard vanKranenburg |
| author_facet | Joyshree Ganguly Maria Martin‐Pascual Richard vanKranenburg |
| author_sort | Joyshree Ganguly |
| collection | DOAJ |
| description | Summary Hungateiclostridium thermocellum DSM 1313 has biotechnological potential as a whole‐cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been hampered due to the lack of simple and high‐throughput genome engineering tools. Recently in our research group, a thermophilic bacterial CRISPR–Cas9‐based system has been developed as a transcriptional suppression tool for regulation of gene expression. We applied ThermoCas9‐based CRISPR interference (CRISPRi) to repress the H. thermocellum central metabolic lactate dehydrogenase (ldh) and phosphotransacetylase (pta) genes. The effects of repression on target genes were studied based on transcriptional expression and product formation. Single‐guide RNA (sgRNA) under the control of native intergenic 16S/23S rRNA promoter from H. thermocellum directing the ThermodCas9 to the promoter region of both pta and ldh silencing transformants reduced expression up to 67% and 62% respectively. This resulted in 24% and 17% decrease in lactate and acetate production, correspondingly. Hence, the CRISPRi approach for H. thermocellum to downregulate metabolic genes can be used for remodelling of metabolic pathways without the requisite for genome engineering. These data established for the first time the feasibility of employing CRISPRi‐mediated gene repression of metabolic genes in H. thermocellum DSM 1313. |
| first_indexed | 2024-12-10T08:49:06Z |
| format | Article |
| id | doaj.art-20cc5762cab740968b37411d413d5ebf |
| institution | Directory Open Access Journal |
| issn | 1751-7915 |
| language | English |
| last_indexed | 2024-12-10T08:49:06Z |
| publishDate | 2020-03-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj.art-20cc5762cab740968b37411d413d5ebf2022-12-22T01:55:38ZengWileyMicrobial Biotechnology1751-79152020-03-0113233934910.1111/1751-7915.13516CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313Joyshree Ganguly0Maria Martin‐Pascual1Richard vanKranenburg2Corbion Arkelsedijk 46 4206 AC Gorinchem The NetherlandsLaboratory of Microbiology Wageningen University Stippeneng 4 6708 WE Wageningen The NetherlandsCorbion Arkelsedijk 46 4206 AC Gorinchem The NetherlandsSummary Hungateiclostridium thermocellum DSM 1313 has biotechnological potential as a whole‐cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been hampered due to the lack of simple and high‐throughput genome engineering tools. Recently in our research group, a thermophilic bacterial CRISPR–Cas9‐based system has been developed as a transcriptional suppression tool for regulation of gene expression. We applied ThermoCas9‐based CRISPR interference (CRISPRi) to repress the H. thermocellum central metabolic lactate dehydrogenase (ldh) and phosphotransacetylase (pta) genes. The effects of repression on target genes were studied based on transcriptional expression and product formation. Single‐guide RNA (sgRNA) under the control of native intergenic 16S/23S rRNA promoter from H. thermocellum directing the ThermodCas9 to the promoter region of both pta and ldh silencing transformants reduced expression up to 67% and 62% respectively. This resulted in 24% and 17% decrease in lactate and acetate production, correspondingly. Hence, the CRISPRi approach for H. thermocellum to downregulate metabolic genes can be used for remodelling of metabolic pathways without the requisite for genome engineering. These data established for the first time the feasibility of employing CRISPRi‐mediated gene repression of metabolic genes in H. thermocellum DSM 1313.https://doi.org/10.1111/1751-7915.13516 |
| spellingShingle | Joyshree Ganguly Maria Martin‐Pascual Richard vanKranenburg CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313 Microbial Biotechnology |
| title | CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313 |
| title_full | CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313 |
| title_fullStr | CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313 |
| title_full_unstemmed | CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313 |
| title_short | CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313 |
| title_sort | crispr interference crispri as transcriptional repression tool for hungateiclostridium thermocellum dsm 1313 |
| url | https://doi.org/10.1111/1751-7915.13516 |
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