Fungal food spoilage often starts with a contamination with spores. Experimental data strongly indicate the existence of subpopulations of spores with different levels of resistance to preservation... Show moreFungal food spoilage often starts with a contamination with spores. Experimental data strongly indicate the existence of subpopulations of spores with different levels of resistance to preservation methods. In this thesis, the extent of this heterogeneity and the underlying mechanisms using fungal model systems is studied. The role of the genetic background, environmental conditions and the developmental state of the spores were studied, using quantitative imaging, genome and RNA/protein sequencing as well as functional gene analysis. The role of transcription factors in weak acid stress resistance of Aspergillus niger is described. Next, heat resistance of fungal spores of three food spoilage species was quantified and compared. The genomes of Aspergillus niger strains were sequenced and compared revealing the existence of a possible sexual cycle. Melanin of fungal spores impacts UV-C resistance, but not heat resistance and a functional CRISPR/Cas9 genome editing system for Paecilomyces variotii and Penicillium roqueforti is described. Older spores are more heat resistant than younger spores, which can be contributed to differences in compatible solute composition. Additionally, a high cultivation temperature results in fungal spores with high heat resistance, possibly due to heat shock proteins. Show less
Aspergillus niger is an important industrial producer of organic acids and enzymes producing large amounts of spent fungal biomass. In the European Research Area Industrial Biotechnology (ERA-IB)... Show moreAspergillus niger is an important industrial producer of organic acids and enzymes producing large amounts of spent fungal biomass. In the European Research Area Industrial Biotechnology (ERA-IB) funded project, we effectively aimed to improve the composition of post-fermentation fungal biomass for extraction of the value-added product chitosan as a derivative of cell wall chitin (FunChi). As chitin/chitosan is not encountered in plant or human tissue, it often acts as an elicitor to plant and animal immune responses in order to fight off possible impending fungal infections. The application of both chitin and chitosan oligomers have been shown to prime plants against infection. This thesis discusses the identification of genes that are important for chitin deposition in the cell wall of A. niger. In addition, the work described here also investigates the genes that facilitate chitin cross-linking to the cell wall. The relevance of all findings are discussed in relation to both the improvement of chitin extraction from post-fermentation biomass and to the integrity of the fungal cell wall. Show less
Pectin is a plant cell wall polysaccharide made of mainly D-galacturonic acid (GA) subunits. The potency of the filamentous fungus Aspergillus niger to naturally secrete high amounts of pectinases... Show morePectin is a plant cell wall polysaccharide made of mainly D-galacturonic acid (GA) subunits. The potency of the filamentous fungus Aspergillus niger to naturally secrete high amounts of pectinases to degrade pectin has been utilized for the industrial production of pectinases. Industrially produced pectinases by A. niger are subsequently used mainly in the food industry, and for the hydrolysis of plant biomass to produce renewable energy. In this thesis, the key players in the transcriptional regulatory system of pectinases in A. niger, such as the transcriptional activator GaaR, the repressor GaaX and the physiological inducer 2-keto-3-deoxy-L-galactonate, are discovered. Several approaches to exploit this system for increased or constitutive expression of the genes encoding pectinases are also presented, such as overexpression and constitutive activation of gaaR, accumulation of 2-keto-3-deoxy-L-galactonate, and deletion of gaaX or creA, the main transcriptional repressor involved in CCR. Show less
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 produces a wide variety of carbohydrate hydrolytic enzymes which have potential applications in the baking, starch, textile, food and feed industries. The goal of this thesis is... Show moreAspergillus niger produces a wide variety of carbohydrate hydrolytic enzymes which have potential applications in the baking, starch, textile, food and feed industries. The goal of this thesis is to unravel the molecular mechanisms of starch and inulin modifying network of A. niger, in order to improve the enzyme production and substrate utilization as well as to find novel enzyme activities. The research described in this thesis shows how we identify genes which encode enzymes involved in starch and inulin catabolism, how they are transcriptionally regulated, as well as how the expressed enzymes react to the carbon sources and what are their physiological roles in A. niger. Important findings are outlined as follows. We have identified inulin pathway activator InuR. The activation of InuR is thought to be triggered by the pathway inducer, sucrose or its derivative, but not previously known fructose. The activated InuR induces the expression of genes encoding inulin modifying enzymes and related sugar transporters in inulin metabolism. In addition, using the tightly regulated inuE promoter combined with racA(G12V) and GFP reporter genes, we set up a novel screening method for isolation of mutants involved in inulin signaling pathway. 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
