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

剪切力对黑曲霉形态发生基因网络、细胞完整性、微观和宏观形态以及细胞内和细胞外产物形成率的影响

• 批准号: 315305620
• 负责人: Professor Dr.-Ing. Rudibert King
• 金额: --
• 依托单位: Fachgebiet Angewandte und Molekulare Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/315305620?language=en
• 关键词: effect; shear; forces; morphogenetic; gene

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.

由于其高分泌潜力，丝状真菌如尼日尔曲霉在生物技术中被广泛用作生产蛋白质和酶的细胞工厂。最近，蛋白质分泌仅通过囊泡运输到菌丝顶端的教条被驳斥。越来越多的证据表明丝状真菌也沿着菌丝分泌蛋白质。菌丝伸长和分枝形成菌丝生长的基础，并且与分泌途径以及细胞壁生物合成紧密相关。细胞壁的生物合成又依赖于细胞外剪切应力条件。剪切应力越高，由于几丁质沉积增加，细胞壁越厚。然而，细胞壁越硬，能沿菌丝沿着分泌的蛋白质就越少。该提案的目的是对受到机械应力条件的尼日尔曲霉进行定性和定量分析。多尺度视图-从基因组，转录组，蛋白质组到亚细胞和反应器水平-将使我们能够全面了解生产条件下尼日尔的机械应力感知和弹性。我们将验证两个中心假设，以便为A的合理菌株改良提供线索。尼日尔用于增加产物形成。我们的假设是：1） 蛋白质的顶端分泌受到囊泡可用性的限制。在高剪切应力条件下，囊泡主要用于细胞壁物质的运输，以加强真菌细胞壁。 在低剪切应力条件下，更多的囊泡可用于分泌蛋白的运输。此外，较不坚硬的细胞壁将允许更多的蛋白质沿着菌丝沿着分泌，从而提高比分泌速率。该项目涉及三个研究小组的跨学科合作。研究组1将产生转基因A。尼日尔菌株具有不同的剪切应力抗性和分支模式，并将通过转录组学和共聚焦显微镜分析这些菌株中的蛋白质分泌。第二研究组将定量分析来自流通式生长室的不稳定和局部分布的显微镜数据，以建立菌丝伸长的数学生长模型。