Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9

Joosu Kuivanen (Corresponding Author), Jasmin Wang, Peter Richard

Research output: Contribution to journalArticleScientificpeer-review

35 Citations (Scopus)

Abstract

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.
Original languageEnglish
Article number210
JournalMicrobial Cell Factories
Volume15
DOIs
Publication statusPublished - 2016
MoE publication typeA1 Journal article-refereed

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Clustered Regularly Interspaced Short Palindromic Repeats
RNA Sequence Analysis
Aspergillus niger
Aspergillus
RNA
uronate dehydrogenase
Acids
Metabolic engineering
Metabolic Engineering
Fungi
Genes
Technology
Oxidation
Dicarboxylic Acids
galactaric acid
Gene Deletion
Biomass
Hydrolysis
galacturonic acid

Keywords

  • aspergillus niger
  • metabolic engineering
  • CRISPR
  • pectin
  • d-galacturonic acid
  • galactaric acid
  • mucic acid
  • uronate dehydrogenase

Cite this

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title = "Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9",
abstract = "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.",
keywords = "aspergillus niger, metabolic engineering, CRISPR, pectin, d-galacturonic acid, galactaric acid, mucic acid, uronate dehydrogenase",
author = "Joosu Kuivanen and Jasmin Wang and Peter Richard",
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journal = "Microbial Cell Factories",
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Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9. / Kuivanen, Joosu (Corresponding Author); Wang, Jasmin; Richard, Peter.

In: Microbial Cell Factories, Vol. 15, 210, 2016.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9

AU - Kuivanen, Joosu

AU - Wang, Jasmin

AU - Richard, Peter

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

KW - aspergillus niger

KW - metabolic engineering

KW - CRISPR

KW - pectin

KW - d-galacturonic acid

KW - galactaric acid

KW - mucic acid

KW - uronate dehydrogenase

U2 - 10.1186/s12934-016-0613-5

DO - 10.1186/s12934-016-0613-5

M3 - Article

VL - 15

JO - Microbial Cell Factories

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SN - 1475-2859

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ER -