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Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9
BACKGROUND: meso-Galactaric acid is a dicarboxylic acid that can be produced by the oxidation of d-galacturonic acid, the main constituent of pectin. Mould strains can be engineered to perform this oxidation by expressing the bacterial enzyme uronate dehydrogenase. In addition, the endogenous pathwa...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5153877/ https://www.ncbi.nlm.nih.gov/pubmed/27955649 http://dx.doi.org/10.1186/s12934-016-0613-5 |
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author | Kuivanen, Joosu Wang, Y.-M. Jasmin Richard, Peter |
author_facet | Kuivanen, Joosu Wang, Y.-M. Jasmin Richard, Peter |
author_sort | Kuivanen, Joosu |
collection | PubMed |
description | BACKGROUND: meso-Galactaric acid is a dicarboxylic acid that can be produced by the oxidation of d-galacturonic acid, the main constituent of pectin. Mould strains can be engineered to perform this oxidation by expressing the bacterial enzyme uronate dehydrogenase. In addition, the endogenous pathway for d-galacturonic acid catabolism has to be inactivated. The filamentous fungus Aspergillus niger would be a suitable strain for galactaric acid production since it is efficient in pectin hydrolysis, however, it is catabolizing the resulting galactaric acid via an unknown catabolic pathway. RESULTS: In this study, a transcriptomics approach was used to identify genes involved in galactaric acid catabolism. Several genes were deleted using CRISPR/Cas9 together with in vitro synthesized sgRNA. As a result, galactaric acid catabolism was disrupted. An engineered A. niger strain combining the disrupted galactaric and d-galacturonic acid catabolism with an expression of a heterologous uronate dehydrogenase produced galactaric acid from d-galacturonic acid. The resulting strain was also converting pectin-rich biomass to galactaric acid in a consolidated bioprocess. CONCLUSIONS: In the present study, we demonstrated the use of CRISPR/Cas9 mediated gene deletion technology in A. niger in an metabolic engineering application. As a result, a strain for the efficient production of galactaric acid from d-galacturonic acid was generated. The present study highlights the usefulness of CRISPR/Cas9 technology in the metabolic engineering of filamentous fungi. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0613-5) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5153877 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-51538772016-12-20 Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9 Kuivanen, Joosu Wang, Y.-M. Jasmin Richard, Peter Microb Cell Fact Research BACKGROUND: meso-Galactaric acid is a dicarboxylic acid that can be produced by the oxidation of d-galacturonic acid, the main constituent of pectin. Mould strains can be engineered to perform this oxidation by expressing the bacterial enzyme uronate dehydrogenase. In addition, the endogenous pathway for d-galacturonic acid catabolism has to be inactivated. The filamentous fungus Aspergillus niger would be a suitable strain for galactaric acid production since it is efficient in pectin hydrolysis, however, it is catabolizing the resulting galactaric acid via an unknown catabolic pathway. RESULTS: In this study, a transcriptomics approach was used to identify genes involved in galactaric acid catabolism. Several genes were deleted using CRISPR/Cas9 together with in vitro synthesized sgRNA. As a result, galactaric acid catabolism was disrupted. An engineered A. niger strain combining the disrupted galactaric and d-galacturonic acid catabolism with an expression of a heterologous uronate dehydrogenase produced galactaric acid from d-galacturonic acid. The resulting strain was also converting pectin-rich biomass to galactaric acid in a consolidated bioprocess. CONCLUSIONS: In the present study, we demonstrated the use of CRISPR/Cas9 mediated gene deletion technology in A. niger in an metabolic engineering application. As a result, a strain for the efficient production of galactaric acid from d-galacturonic acid was generated. The present study highlights the usefulness of CRISPR/Cas9 technology in the metabolic engineering of filamentous fungi. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0613-5) contains supplementary material, which is available to authorized users. BioMed Central 2016-12-12 /pmc/articles/PMC5153877/ /pubmed/27955649 http://dx.doi.org/10.1186/s12934-016-0613-5 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Kuivanen, Joosu Wang, Y.-M. Jasmin Richard, Peter Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9 |
title | Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9 |
title_full | Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9 |
title_fullStr | Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9 |
title_full_unstemmed | Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9 |
title_short | Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9 |
title_sort | engineering aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using rna sequencing and crispr/cas9 |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5153877/ https://www.ncbi.nlm.nih.gov/pubmed/27955649 http://dx.doi.org/10.1186/s12934-016-0613-5 |
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