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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...

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Autores principales: Kuivanen, Joosu, Wang, Y.-M. Jasmin, Richard, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
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.
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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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