title: Rewiring a secondary metabolite pathway towards itaconic acid production in Aspergillus niger.
abstract: BACKGROUND
  The industrially relevant filamentous fungus Aspergillus niger is widely used in industry for its secretion capabilities of enzymes and organic acids. Biotechnologically produced organic acids promise to be an attractive alternative for the chemical industry to replace petrochemicals. Itaconic acid (IA) has been identified as one of the top twelve building block chemicals which have high potential to be produced by biotechnological means. The IA biosynthesis cluster (cadA, mttA and mfsA) has been elucidated in its natural producer Aspergillus terreus and transferred to A. niger to enable IA production. Here we report the rewiring of a secondary metabolite pathway towards further improved IA production through the overexpression of a putative cytosolic citrate synthase citB in a A. niger strain carrying the IA biosynthesis cluster.
  
  
  RESULTS
  We have previously shown that expression of cadA from A. terreus results in itaconic acid production in A. niger AB1.13, albeit at low levels. This low-level production is boosted fivefold by the overexpression of mttA and mfsA in itaconic acid producing AB1.13 CAD background strains. Controlled batch cultivations with AB1.13 CAD + MFS + MTT strains showed increased production of itaconic acid compared with AB1.13 CAD strain. Moreover, preliminary RNA-Seq analysis of an itaconic acid producing AB1.13 CAD strain has led to the identification of the putative cytosolic citrate synthase citB which was induced in an IA producing strain. We have overexpressed citB in a AB1.13 CAD + MFS + MTT strain and by doing so hypothesize to have targeted itaconic acid production to the cytosolic compartment. By overexpressing citB in AB1.13 CAD + MFS + MTT strains in controlled batch cultivations we have achieved highly increased titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h while no CA was produced.
  
  
  CONCLUSIONS
  Expression of the IA biosynthesis cluster in Aspergillus niger AB1.13 strain enables IA production. Moreover, in the AB1.13 CAD strain IA production resulted in overexpression of a putative cytosolic citrate synthase citB. Upon overexpression of citB we have achieved titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h in controlled batch cultivations. By overexpressing citB we have also diminished side product formation and optimized the production pathway towards IA.
authors.affiliation: Dutch DNA Biotech B.V, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands. abeer.hossain@ddna-biotech.com.|Molecular Biology and Microbial Food Safety, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands. abeer.hossain@ddna-biotech.com.
authors.forename: Abeer H
authors.initials: AH
authors.lastname: Hossain
authors.affiliation: Microbiology and Systems Biology, TNO, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
authors.forename: An
authors.initials: A
authors.lastname: Li
authors.affiliation: Microbiology and Systems Biology, TNO, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
authors.forename: Anja
authors.initials: A
authors.lastname: Brickwedde
authors.affiliation: Dutch DNA Biotech B.V, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
authors.forename: Lars
authors.initials: L
authors.lastname: Wilms
authors.affiliation: Microbiology and Systems Biology, TNO, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
authors.forename: Martien
authors.initials: M
authors.lastname: Caspers
authors.affiliation: Dutch DNA Biotech B.V, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
authors.forename: Karin
authors.initials: K
authors.lastname: Overkamp
authors.affiliation: Dutch DNA Biotech B.V, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Punt
mesh_terms: D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism*; D002950:Citrate (si)-Synthase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005285:Fermentation; D018014:Gene Transfer Techniques; D053858:Metabolic Networks and Pathways / Q000235:genetics; D013386:Succinates / Q000378:metabolism*
chemical_list: D013386:Succinates; D002950:Citrate (si)-Synthase; C005229:itaconic acid
keywords: Citric acid; Controlled-batch cultivation; Itaconic acid; Secondary metabolite; citB