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.
authors.affiliation: VTT Technical Research Centre of Finland Ltd, P. O. Box 1000, FI-02044, Espoo, Finland. joosu.kuivanen@vtt.fi.
authors.forename: Joosu
authors.initials: J
authors.lastname: Kuivanen
authors.affiliation: VTT Technical Research Centre of Finland Ltd, P. O. Box 1000, FI-02044, Espoo, Finland.
authors.forename: Y-M Jasmin
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: VTT Technical Research Centre of Finland Ltd, P. O. Box 1000, FI-02044, Espoo, Finland.
authors.forename: Peter
authors.initials: P
authors.lastname: Richard
mesh_terms: D001234:Aspergillus niger / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D060847:Metabolic Engineering / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D013400:Sugar Acids; C000090:galactaric acid
keywords: Aspergillus niger; CRISPR; D-galacturonic acid; Galactaric acid; Metabolic engineering; Mucic acid; Pectin; Uronate dehydrogenase