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

由于其高分泌能力，丝状真菌如黑曲霉被广泛应用于生物技术中，作为生产蛋白质和酶的细胞工厂。最近，蛋白质分泌只通过小泡向菌丝尖端运输的说法被驳斥。越来越多的证据表明，丝状真菌也会沿着菌丝分泌蛋白质。菌丝伸长和分枝是菌丝生长的基础，与分泌途径和细胞壁生物合成密切相关。细胞壁的生物合成又依赖于细胞外的剪应力条件。剪切应力越大，由于几丁质沉积增加，细胞壁越厚。然而，细胞壁越坚硬，沿菌丝分泌的蛋白质就越少。该提案的目的是对机械应力条件下的黑曲霉进行定性和定量分析。从基因组、转录组、蛋白质组到亚细胞和反应器水平的多尺度视角将使我们能够全面了解黑曲霉在生产条件下的机械应力感知和弹性。我们将验证两个中心假设，以便为合理改良黑曲霉增加产物形成而产生线索。我们的假设是：1)蛋白质的顶端分泌受到小泡可获得性的限制。在高切应力条件下，小泡主要用于运输细胞壁物质，以加强真菌的细胞壁。2)在低切应力条件下，更多的小泡可用于分泌蛋白的运输。此外，不那么坚硬的细胞壁将允许沿菌丝分泌更多的蛋白质，从而增加特定的分泌率。该项目涉及三个研究小组的跨学科合作。研究一组将产生具有不同剪切应力抗性和分枝模式的转基因黑曲霉菌株，并将通过转录组和共聚焦显微镜分析这些菌株的蛋白质分泌。研究小组2将对来自流动生长室的现场和局部分布的微观数据进行定量分析，以开发菌丝伸长的数学生长模型。

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.