title: Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures.
abstract: The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.
authors.affiliation: Microbiology and Kluyver Centre for Genomics of Industrial Fermentation, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Charissa
authors.initials: C
authors.lastname: de Bekker
authors.forename: G Jerre
authors.initials: GJ
authors.lastname: van Veluw
authors.forename: Arman
authors.initials: A
authors.lastname: Vinck
authors.forename: L Ad
authors.initials: LA
authors.lastname: Wiebenga
authors.forename: Han A B
authors.initials: HA
authors.lastname: Wösten
mesh_terms: D001234:Aspergillus niger / Q000145:classification / Q000166:cytology* / Q000235:genetics* / Q000648:ultrastructure; D019149:Bioreactors; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D005285:Fermentation; D005434:Flow Cytometry; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D025282:Mycelium / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000032:analysis
chemical_list: D049452:Green Fluorescent Proteins; D012313:RNA; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D005087:Glucan 1,4-alpha-Glucosidase