Applications & Publications

Spatial induction of genes encoding secreted proteins in micro-colonies of Aspergillus niger

Aspergillus niger is used by the industry to produce enzymes and metabolites such as citric acid. In liquid cultures, it can grow as a dispersed mycelium or as micro-colonies with a width in the micrometer to millimeter range. Here, it was assessed whether expression of genes encoding secreted enzymes depends on mycelium morphology. To this end, expression of the reporter gene gfp from the promoters of the glucoamylase gene glaA, the feruloyl esterase gene faeA and the α-glucuronidase gene aguA was causally related to micro-colony size within a liquid shaken culture. Data could be fitted by hyperbolic functions, implying that the genes encoding these secreted proteins are expressed in a shell at the periphery of the micro-colony. The presence of such a shell was confirmed by confocal microscopy. Modelling predicted that the width of these zones was 13 to 156 µm depending on growth medium and micro-colony diameter. Together, data indicate that the highest productive micro-colonies are those colonies that have a radius ≤ the width of the peripheral expression zone.

Mixed colonies of Aspergillus niger and Aspergillus oryzae cooperatively degrading wheat bran

Heterogeneity in liquid shaken cultures of Aspergillus niger inoculated with melanised conidia or conidia of pigmentation mutant

1) Microbiology and Kluyver Centre for Genomics of Industrial Fermentations, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 2) Biomolecular Imaging, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 3) Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands; 4) Department of BioMedical Engineering, University Medical Center Groningen and University of Groningen,Hanzeplein 1, 9713 AV Groningen, The Netherlands; 5CBS-KNAW Fungal Diversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

*Correspondence: H.A.B. Wösten, h.a.b.wosten@uu.nl

Heterogeneity of Aspergillus niger in micro-colonies in Liquid Shaken Cultures.

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.

Improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other fungi

¹ Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ² School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, Hong Kong SAR, China ³ Ministry of Agriculture Key Laboratory of Subtropical Agro-biological Disaster and Management, Fujian Agriculture and Forestry University, Fuzhou 350002, China ⁴ CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands ⁵ Kluyver Centre for Industrial Fermentation, Utrecht University, Utrecht, The Netherlands