The research described in this thesis aims to get more fundamental insights in the molecular mechanisms used by Aspergillus niger in relation to control morphology and protein secretion. Knowledge... Show moreThe research described in this thesis aims to get more fundamental insights in the molecular mechanisms used by Aspergillus niger in relation to control morphology and protein secretion. Knowledge on these two aspects is highly relevant to further optimization of A.niger as a cell factory Show less
Itaconic acid (methylene succinic acid) is a white crystalline unsaturated C5 dicarboxylic acid. Because of its specific favorable properties and the unique structure, itaconic acid is used as... Show moreItaconic acid (methylene succinic acid) is a white crystalline unsaturated C5 dicarboxylic acid. Because of its specific favorable properties and the unique structure, itaconic acid is used as monomer or co-monomer in the manufacture of plastics, resins, synthetic fibers, paints, surfactant and elastomers etc. Besides, in 2004 it was selected by the Department of Energy in the US as one of the 12 building block chemicals that are the most interesting to be produced by industrial biotechnology. Currently itaconic acid is produced commercially by Aspergillus terreus via fungal fermentation. Due to its limited availability, the use of itaconic acid is restricted. To increase itaconic acid production levels and make it economically available, Aspergillus niger is chosen as the production host strain due to its flexibility as a production platform. Besides, A. niger accumulates organic acids on a wide range of substrat es under various environmental conditions. Most important, A. niger produces large amounts of citric acid (360 g/L) which in A. terreus actually is a precursor in the biosynthetic pathway towards itaconic acid. This thesis describes the research carried out for producing itaconic acid in A. niger, and illustrates research approaches of organic acids production in fungi in general Show less
In this thesis we have focused our studies towards a better understanding of different processes involved in the protein secretion pathway that might act as bottlenecks for homologous and... Show moreIn this thesis we have focused our studies towards a better understanding of different processes involved in the protein secretion pathway that might act as bottlenecks for homologous and heterologous protein production. We have given particular attention to the molecular mechanisms of folding and quality control that take place in the ER, in order to be able to improve and develop new strategies for heterologous protein production by A. niger. Show less
Accumulation of unfolded proteins within the lumen of the ER results in secretion stress in eukaryotic organisms. The industrial important filamentous fungus Aspergillus niger, responds to this... Show moreAccumulation of unfolded proteins within the lumen of the ER results in secretion stress in eukaryotic organisms. The industrial important filamentous fungus Aspergillus niger, responds to this stress by activating a transcriptional activator. This transcriptional activator, HacA, up- regulates its target genes by binding to UPR elements present in the promoters of those genes. These target genes include foldases and chaperones that assist protein folding in the ER. The thesis describes regulation of the A. niger UPR and activation of HacA, which involves a high degree of auto-regulation. 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
Aspergillus niger is a cosmopolitan fungus and its spores can be found in air and soil worldwide. This saprophyte is used in food biotechnology for the production of proteins, mainly enzymes and... Show moreAspergillus niger is a cosmopolitan fungus and its spores can be found in air and soil worldwide. This saprophyte is used in food biotechnology for the production of proteins, mainly enzymes and for the production of organic acids. In the production of proteins, several problems are encountered such as repressed gene-expression, morphologic and genetic instability and undesired metabolite accumulation. To find solutions to these problems, often mutagenesis and mitotic recombination have been used, as described in this thesis. These mutations cannot simply be recombined by crosses, as Aspergillus niger is an asexual fungus. An alternative way to recombine mutations is via the parasexual cycle, also called mitotic recombination. To obtain more information of its possibilities we studied mitotic recombination of genetic markers on chromosome III. Recently the genome sequence of A. niger has been determined and this information has been linked to the genetic map. By improving the genetic map of chromosome III we were able to study crossing-over of linked markers and found that these cross-over events occurred predominantly in a small part of the chromosome. This mitotic recombination hotspot has some interesting physical features, but it remains uncertain if and which of them is responsible for the increase in crossing-over. Show less
Antimicrobial compounds have many applications, in medicines, food, agriculture, livestock, textiles, paints, and wood protectants. Microorganisms resistant to most antibiotics are rapidly... Show moreAntimicrobial compounds have many applications, in medicines, food, agriculture, livestock, textiles, paints, and wood protectants. Microorganisms resistant to most antibiotics are rapidly spreading. Consequently there is an urgent and continuous need for novel antimicrobial compounds. Most antibiotics have been developed from microorganisms. Plants also represent an important source for finding novel antimicrobial compounds, as plants in their permanent fight with microorganisms in their environment produce a wide spectrum of compounds with antimicrobial activity. This thesis focuses specifically on abundantly available plant sources, with the idea that plants already processed in the agricultural industry might be the source of antimicrobial compounds which could add extra value to these crops. It was shown in this project that Cannabis sativa (Cannabis), Humulus lupulus (Hop) and Tectona grandis (Teak) extracts display antimicrobial activities in general screening methods. Teak sawdust extract also showed antifungal activities against Aspergillus niger and four strains of wood rot fungi. Several active compounds were isolated. Deoxylapachol, for example isolated from teak sawdust extract, inhibits wood rot fungi by induction of fungal cell wall stress and inhibition of cellulase activity. This project shows __the proof of concept__ for the hypothesis that raw and waste materials from common agri/horticultural industry processes can serve as sources for new biologically active compounds. Show less
