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Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes
Pseudoclostridium thermosuccinogenes is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a platform organism. However, exploitation of P. thermosuccinogenes has been limited partly due to the genetic inaccessibility...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9044749/ https://www.ncbi.nlm.nih.gov/pubmed/35456750 http://dx.doi.org/10.3390/microorganisms10040698 |
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author | Ganguly, Joyshree Martin-Pascual, Maria Montiel González, Diego Bulut, Alkan Vermeulen, Bram Tjalma, Ivo Vidaki, Athina van Kranenburg, Richard |
author_facet | Ganguly, Joyshree Martin-Pascual, Maria Montiel González, Diego Bulut, Alkan Vermeulen, Bram Tjalma, Ivo Vidaki, Athina van Kranenburg, Richard |
author_sort | Ganguly, Joyshree |
collection | PubMed |
description | Pseudoclostridium thermosuccinogenes is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a platform organism. However, exploitation of P. thermosuccinogenes has been limited partly due to the genetic inaccessibility and lack of genome engineering tools. In this study, we established the genetic accessibility for P. thermosuccinogenes DSM 5809. By overcoming restriction barriers, transformation efficiencies of 10(2) CFU/µg plasmid DNA were achieved. To this end, the plasmid DNA was methylated in vivo when transformed into an engineered E. coli HST04 strain expressing three native methylation systems of the thermophile. This protocol was used to introduce a ThermodCas9-based CRISPRi tool targeting the gene encoding malic enzyme in P. thermosuccinogenes, demonstrating the principle of gene silencing. This resulted in 75% downregulation of its expression and had an impact on the strain’s fermentation profile. Although the details of the functioning of the restriction modification systems require further study, in vivo methylation can already be applied to improve transformation efficiency of P. thermosuccinogenes. Making use of the ThermodCas9-based CRISPRi, this is the first example demonstrating that genetic engineering in P. thermosuccinogenes is feasible and establishing the way for metabolic engineering of this bacterium. |
format | Online Article Text |
id | pubmed-9044749 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-90447492022-04-28 Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes Ganguly, Joyshree Martin-Pascual, Maria Montiel González, Diego Bulut, Alkan Vermeulen, Bram Tjalma, Ivo Vidaki, Athina van Kranenburg, Richard Microorganisms Article Pseudoclostridium thermosuccinogenes is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a platform organism. However, exploitation of P. thermosuccinogenes has been limited partly due to the genetic inaccessibility and lack of genome engineering tools. In this study, we established the genetic accessibility for P. thermosuccinogenes DSM 5809. By overcoming restriction barriers, transformation efficiencies of 10(2) CFU/µg plasmid DNA were achieved. To this end, the plasmid DNA was methylated in vivo when transformed into an engineered E. coli HST04 strain expressing three native methylation systems of the thermophile. This protocol was used to introduce a ThermodCas9-based CRISPRi tool targeting the gene encoding malic enzyme in P. thermosuccinogenes, demonstrating the principle of gene silencing. This resulted in 75% downregulation of its expression and had an impact on the strain’s fermentation profile. Although the details of the functioning of the restriction modification systems require further study, in vivo methylation can already be applied to improve transformation efficiency of P. thermosuccinogenes. Making use of the ThermodCas9-based CRISPRi, this is the first example demonstrating that genetic engineering in P. thermosuccinogenes is feasible and establishing the way for metabolic engineering of this bacterium. MDPI 2022-03-24 /pmc/articles/PMC9044749/ /pubmed/35456750 http://dx.doi.org/10.3390/microorganisms10040698 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Ganguly, Joyshree Martin-Pascual, Maria Montiel González, Diego Bulut, Alkan Vermeulen, Bram Tjalma, Ivo Vidaki, Athina van Kranenburg, Richard Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes |
title | Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes |
title_full | Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes |
title_fullStr | Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes |
title_full_unstemmed | Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes |
title_short | Breaking the Restriction Barriers and Applying CRISPRi as a Gene Silencing Tool in Pseudoclostridium thermosuccinogenes |
title_sort | breaking the restriction barriers and applying crispri as a gene silencing tool in pseudoclostridium thermosuccinogenes |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9044749/ https://www.ncbi.nlm.nih.gov/pubmed/35456750 http://dx.doi.org/10.3390/microorganisms10040698 |
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