Streptomycetes are Gram-positive multicellular soil-dwelling bacteria which are commercially used as natural producers of antibiotics, anticancer agents and immunosuppressants, as well as many... Show moreStreptomycetes are Gram-positive multicellular soil-dwelling bacteria which are commercially used as natural producers of antibiotics, anticancer agents and immunosuppressants, as well as many industrial enzymes (Hopwood 2007). Similarly to fungi, they carry out a complex developmental life cycle, forming highly structured multicellular colonies composed of physiologically distinct hyphae (Miguelez et al. 2000). A major scientific challenge lies in understanding how growth parameters are controlled in response to nutritional conditions, and how this affects the efficiency of production and secretion of proteins and antibiotics. The work presented in this thesis therefore aims to arrive at a better understanding of Streptomyces morphogenesis and development, and how these processes link to productivity. Studies performed include the analysis of several novel coiled-coil proteins and their effect on morphogenesis an d division, as well as fluorescent live imaging to better understand and localize dynamic secretion systems. Cryo-correlative light and electron microscopy, and specifically tomography, was performed to image vegetative cell division and its effect on development within a pellet. Combining the multi-scale information gained, a structured morphological model was created to provide a framework for rational design of Streptomyces sp. and provide a test drive for the fermentation process. Show less
Filamentous fungi are multicellular eukaryotic organisms, which represent a separate taxonomic group organisms within the fungal kingdom, apart from the yeasts. These fungi always need a substrate... Show moreFilamentous fungi are multicellular eukaryotic organisms, which represent a separate taxonomic group organisms within the fungal kingdom, apart from the yeasts. These fungi always need a substrate to grow on, this can be living or dead material. Fungi possess the capacity to secrete high levels of enzymes. Because of this specific property, fungi are already used for centuries as miniature factories for the production of extracellular proteins. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of, heterologous proteins. This low protein yield is likely to be caused by impaired secretion of the heterologous protein. Evidence support the idea that a bottleneck for protein production is post-translational, possibly within the protein secretion pathway. To be able to improve heterologous protein secretion in fungi the (molecular) mechanism(s) of the protein secretion pathway was studied and resulted in this thesis. In Chapter 1 the knowledge about the secretory pathway in the yeast S. cerevisiae has been used as starting point to discuss and review different aspects of protein secretion in filamentous fungi. Special focus is on the comparison of the presence and function of secretion related small GTPases in yeasts, mammalian cells and filamentous fungi. Chapter 2 describes the visualisation of different cell organelles from A. niger using GFP-reporter proteins. To target GFP to a specific organelle, the GFP was fused to an organelle specific protein, or part of such a protein. In this way it was possible to visualize, nuclei, the endoplasmic reticulum (ER) and vacuoles. In addition, by fusing GFP to a protein that is efficiently secreted (glucoamylase), also the protein secretion process could be visualised. Chapter 3 describes the identification of several small GTPases in A. niger. The function of one of them, srgA, has been studied in more detail. In Chapter 4 the in depth functional characterisation of a second secretion related GTPase from A. niger named srgC is described. Here it is shown that this secretion related GTPase is specifically important for vacuolar biosynthesis which is visualized by the GFP-reporters described in chapter 2. In Chapter 5 a study on heterologous protein production in A. niger is described. Here a novel screening method is used based on a fusion protein between the well secreted A. niger glucoamylase protein fused with a laccase from Pleurotus ostreatus. With this method laccase hyper-secretion mutants were isolated. Show less
