The effect of shear forces on the morphogenetic gene network, cell integrity, microscopic and macroscopic morphology of Aspergillus niger and on the formation rate of intra- and extracellular products

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Due to their high secretion potential, filamentous fungi such as Aspergillus niger are widely used in biotechnology as cell factories for the production of proteins and enzymes. Most recently, the dogma was refuted that protein secretion only occurs via vesicle transport towards hyphal tips. There is accumulating evidence that filamentous fungi secrete proteins also along hyphae. Hyphal elongation and branching forms the basis for mycelial growth and is tightly linked with the secretory pathway as well as with cell wall biosynthesis. Cell wall biosynthesis in turn is dependent on extracellular shear stress conditions. The higher the shear stress the thicker the cell wall due to increased chitin deposition. However, the more rigid the cell wall the less proteins can be secreted along hyphae.The aim of the proposal is to perform qualitative and quantitative analysis of Aspergillus niger subjected to mechanical stress conditions. A multi-scale view – from the genome, transcriptome, proteome to the subcellular and reactor level – will enable us to holistically understand mechanical stress perception and resilience of Aspergillus niger under production conditions. We will verify two central hypotheses in order to generate leads for rational strain improvement of A. niger for increased product formation. Our hypotheses are:1) Apical secretion of proteins is limited by the availability of vesicles. Under high shear stress conditions, vesicles are mainly used for the transport of cell wall materials to enforce the fungal cell wall.2) Under low shear stress conditions, more vesicles are available for the transport of secretory proteins. In addition, less rigid cell walls will allow higher secretion of proteins along hyphae thus increasing the specific secretion rate.The project involves the interdisciplinary collaboration of three research groups. Research group 1 will generate genetically modified A. niger strains with different shear stress resistance and branching pattern and will analyze protein secretion in these strains by means of transcriptomics and confocal microscopy. Research group 2 will quantitatively analyze instationary and locally distributed microscopic data from a flow-through growth chamber to develop a mathematical growth model for hyphal elongation.