title: Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.
abstract: BACKGROUND
  The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency.
  
  
  RESULTS
  In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein.
  
  
  CONCLUSIONS
  Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.
authors.affiliation: Department Applied and Molecular Microbiology, Institute of Biotechnology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Wanka
authors.affiliation: Department Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
authors.forename: Mark
authors.initials: M
authors.lastname: Arentshorst
authors.affiliation: Department Applied and Molecular Microbiology, Institute of Biotechnology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Cairns
authors.affiliation: Protein Expression, Novo Nordisk, Novo Nordisk Park, 2760, Måløv, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jørgensen
authors.affiliation: Department Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
authors.forename: Arthur F J
authors.initials: AF
authors.lastname: Ram
authors.affiliation: Department Applied and Molecular Microbiology, Institute of Biotechnology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355, Berlin, Germany. vera.meyer@tu-berlin.de.
authors.forename: Vera
authors.initials: V
authors.lastname: Meyer
mesh_terms: D021301:Antifreeze Proteins / Q000235:genetics / Q000378:metabolism; D001234:Aspergillus niger / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D049109:Cell Proliferation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic; D015398:Signal Transduction
chemical_list: D021301:Antifreeze Proteins; D005656:Fungal Proteins; C097609:antifungal protein, Aspergillus; D008156:Luciferases
keywords: Antifungal protein; Aspergillus niger; Hydrophobin; Perfusion cultivation; Promoter; Zero growth rate