Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Induction of prenylated isoflavonoids and stilbenoids in legumes
    Aisyah, S. - \ 2015
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789462574816 - 154
    flavonoïden - stilbenoïden - isoflavonoïden - peulgewassen - glycine max - sojabonen - arachis hypogaea - fylogenetica - phaseolus - lupinus - rhizopus - aspergillus - kwantitatieve analyse - flavonoids - stilbenoids - isoflavonoids - legumes - glycine max - soyabeans - arachis hypogaea - phylogenetics - phaseolus - lupinus - rhizopus - aspergillus - quantitative analysis

    The germination of legume seeds in the presence or absence of stress factors was studied with respect to compositional changes in prenylated isoflavonoids and stilbenoids. Different strategies were applied using (i) different types of legume seed, (ii) different stress factors i.e. biotic, abiotic and their combination, and (iii) different time point of application of the fungus. Mass spectrometric tools to better characterize the position of prenyl groups in the molecules were optimized. Isoflavonoids and stilbenoids appeared more inducible than flavonoids. Fungus was a more effective stress factor than light and wounding. The impact of fungus might be enhanced by combining it with other stress factors; the combination of fungus and light was more promising than that of fungus and wounding. The seeds of various legume species appeared to respond differently towards elicitation by Rhizopus during germination. The kind of molecules induced followed the phylogenetic relationship of the various species, but their amounts induced during germination, alone or combined with elicitation, did not. In terms of quantities of compounds induced, some species such as Glycine max, Phaseolus spp., Lupinus spp. and Arachis hypogaea were more promising than Vigna spp., Lablab purpureus and Psophocarpus tetragonolobus. Moreover, the fact that Rhizopus and Aspergillus could metabolize the stilbenoids induced during the process of simultaneous germination and elicitation of peanut seedlings showed that the type of fungus was a crucial parameter for optimizing accumulation of potentially bioactive compounds.

    Protein redesign by learning from data
    Berg, B.A. van den; Reinders, M.J.T. ; Laan, J.M. van der; Roubos, J.A. ; Ridder, D. de - \ 2014
    Protein Engineering, Design & Selection 27 (2014)9. - ISSN 1741-0126 - p. 281 - 288.
    computational enzyme design - aspergillus - stabilization - optimization - generation - prediction - secretion - hydrolase - peptides - tools
    Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5-45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.
    A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3
    Remoroza, C.A. ; Wagenknecht, M. ; Buchholt, H.C. ; Moerschbacher, B.M. ; Schols, H.A. ; Gruppen, H. - \ 2014
    Carbohydrate Polymers 107 (2014). - ISSN 0144-8617 - p. 85 - 93.
    hairy ramified regions - sugar-beet pectin - erwinia-chrysanthemi 3937 - rhamnogalacturonan acetylesterase - esterification - identification - deacetylation - aspergillus - extraction - oligomers
    A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50 °C. It is stable in the range pH 5.0–7.0 and below 50 °C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, 1H NMR of polymer and LC-HILIC-MSn after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.
    Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants
    Gool, M.P. van; Vansco, I. ; Schols, H.A. ; Toth, K. ; Szakacs, G. ; Gruppen, H. - \ 2011
    Bioresource Technology 102 (2011)10. - ISSN 0960-8524 - p. 6039 - 6047.
    alpha-l-arabinofuranosidases - eucalyptus-globulus labill - wheat-flour arabinoxylan - xylanolytic enzymes - aspergillus - mode - arabinofuranohydrolase - oligosaccharides - purification - esterases
    The efficient degradation of complex xylans needs collaboration of many xylan degrading enzymes. Assays for xylan degrading activities based on reducing sugars or PNP substrates are not indicative for the presence of enzymes able to degrade complex xylans: They do not provide insight into the possible presence of xylanase-accessory enzymes within enzyme mixtures. A new screening method is described, by which specific xylan modifying enzymes can be detected. Fermentation supernatants of 78 different fungal soil isolates grown on wheat straw were analyzed by HPLC and MS. This strategy is powerful in recognizing xylanases, arabinoxylan hydrolases, acetyl xylan esterases and glucuronidases. No fungus produced all enzymes necessary to totally degrade the substrates tested. Some fungi produce high levels of xylanase active against linear xylan, but are unable to degrade complex xylans. Other fungi producing relative low levels of xylanase secrete many useful accessory enzyme component(s).
    An internet-accessible DNA sequence database for identifying fusaria from human and animal infections
    O'Donnell, K. ; Sutton, D.A. ; Rinaldi, M.G. ; Sarver, B.A.J. ; Balajee, S.A. ; Schroers, H.J. ; Summerbell, R.C. ; Robert, V.A.R.G. ; Crous, P.W. ; Zhang, N. ; Aoki, T. ; Jung, K. ; Park, J. ; Lee, Y.H. ; Kang, S. ; Park, B. ; Geiser, D.M. - \ 2010
    Journal of Clinical Microbiology 48 (2010)10. - ISSN 0095-1137 - p. 3708 - 3718.
    solani species complex - vitro antifungal susceptibility - molecular phylogenetic diversity - gene genealogies - fungal pathogens - reproductive isolation - cryptic speciation - contact-lens - identification - aspergillus
    Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1{alpha} (EF-1{alpha}), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate's clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID ( and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center ( Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.
    Macroscopic modelling of solid-state fermentation
    Hoogschagen, M.J. - \ 2007
    Wageningen University. Promotor(en): Hans Tramper, co-promotor(en): Arjen Rinzema. - [S.l.] : S.n. - ISBN 9789085045786 - 120
    fysische modellen - groeimodellen - computersimulatie - aspergillus - tarwe - vast-substraatfermentatie - physical models - growth models - computer simulation - aspergillus - wheat - solid-state fermentation

    Solid-state fermentation is different from the more well known process of liquid fermentation because no free flowing water is present. The technique is primarily used in Asia. Well-known products are the foods tempe, soy sauce and saké. In industrial solid-state fermentation, the substrate usually consists of loose substrate particles, although in research situations agar like substrates are also common. Solid-state fermentations cannot be mixed as easily as liquid fermentations. Because of this, it is difficult to maintain the temperature in the fermentation at an acceptable level and to prevent differences in substrate availability throughout the solid material. An advantage of solid-state fermentation is that the process is cheap, and that products are in some cases easier to separate from the substrate than in liquid. Because of this, the technique is economically interesting. The process has not been studied as extensively as liquid fermentation. This thesis extends the available knowledge by providing several mathematical models for both biological and physical processes that occur in aerated packed beds.

    In aerated packed beds, the metabolic heat that is released in the microbial process is removed by blowing air through the packed material. The effectiveness of the aeration is the result of both the heat uptake capacity of the air itself and of the evaporation of moisture to the air. In fact, the evaporation contributes more to the heat removal than the air itself. A side effect of the evaporation is that the decreasing moisture level in the substrate can become limiting for the microbial process.

    In this thesis, the growth of Aspergillus oryzae and Aspergillus sojae, two related species of fungi, in an aerated packed bed of moist wheat kernels is studied. The study deals with both the microbial and physical aspects of the system.

    Many different types of substrate have been used in studies on solid-state fermentation. Prior to starting the work on the mathematical models, we checked if the fermentation results of A. oryzae on several types of wheat matched, The check was done by matching respiration profiles for several types of wheat and two pretreatment methods. It turned out that considerable differences between the pretreatment methods can exist, which indicated the importance of using the exact same type of substrate and pretreatment in experiments that are to be compared.

    No accurate model description of the microbial aspects of SSF is available yet. Because the focus of the major part of the thesis is on deriving model descriptions for the physical aspects of cooling and drying-out in aerated packed beds, it was decided that using a temperature-response model for the description of heat development would incorporate too many uncertainties in the overall packed-bed model. The heat development in the further studies presented was therefore based on fitted oxygen consumption profiles instead of on modelled microbial growth.

    For the validation of the physical models in this thesis, experiments were carried out in a packed bed of approximately 50 cm height. This packed bed was insulated thermally, and offered the possibility of taking online temperature measurements and sampling the moisture content. The models that were derived to describe the changes in growth conditions in the packed bed in time and space were based on well-known physical relations. All physical models are composed of heat- and mass balances. As described above, the temperature dependence of the fungus was neglected, and the metabolic heat development was incorporated in the balances by means of fitted respiration profiles. This way, inaccuracies in the heat production in the physical model were prevented, allowing the focus on the correct description of heat and mass transfer.

    The first model presented was based on an existing model, which overestimated the drying out of the solid material. This overestimation was due to the assumption of constant saturation of the gas phase with water vapour. The overestimation of the drying out meant that the assumption of vapour saturation needed to be adjusted. Heat and mass transfer coefficients were determined for the substrate involved, and besides this water activity was introduced as a factor that limited the evaporation of water from the substrate. The addition of water activity was of great influence on the model results.

    The insight in the effect of local water activity on the fermentation was the onset for a study on the response of fungi to changing water activity. A system was designed that allowed the dynamic response of the fungus on decreasing water activity to be measured. The experimental set-up was based on isothermal experiments that were slowly dried out by blowing dry air through them, with simultaneous experiments carried out at aw ~ 1 for comparison with the response to the drying out. Considering the fact that all studies on water activity that preceded this approach were based on static and artificial conditions, this set-up is more similar to the actual conditions in a packed-bed fermentation.

    Contrary to the expectations, the system that was dried out showed a decreased fungal growth rate when the water activity in the substrate was still the same as it was in the reference experiment. We checked two possible causes for this phenomenon. Moisture gradients in the particle were ruled out, because these were too small to be able to cause the difference in growth rate. We found that there is most likely a region of very cold substrate material due to wet-bulb cooling. Wet-bulb cooling is a phenomenon in which the evaporation of water from a system to a passing airflow allows the system to cool to temperatures below the temperature of the air. Because we used dry air in our experiment, the effect was too large to be compensated through conduction. An estimated 5% of the bed could not be fermented because of the low temperature. For a successful series of experiments, we need to obtain drying with a 100% constant bed temperature. For such a series, a good comparison of the effect of drying on fungal behaviour will be possible.

    During the experiments in the packed-bed involving Aspergillus oryzae strong shrinkage of the packed-bed occurred because the fungus tied the substrate particles together. Because of the shrinkage, the aeration lost effectivity and the fermentation results were suboptimal. A model was designed to describe the amount of shrinkage, based on the decrease in water content in the bed. The validation of this model could not be performed with A. oryzae, because it was impossible to carry out controlled fermentations with this fungus. Therefore, A. sojae was used, which has the same growth characteristics as A. oryzae, except for the formation of substrate ties. The model on shrinkage offers a good prediction of the shrinkage that is expected with the combined effect of fungal growth and dehydration. 1f in industrial fermentations a different shrinkage pattern is observed, this is an indication that there is channel formation somewhere in the bed. This observation can be than followed by for instance a mixing event, improving the overall performance of the fermentation.

    In the final chapter of this thesis, an overview of the work that could possibly offer further improvements to the present models is given. It was concluded that the modelling of the microbial aspects offer the biggest chances for success in this respect, since this aspect has of yet been modelled less accurately than the physical part.

    Germination of Penicillium paneum Conidia Is Regulated by 1-Octen-3-ol, a Volatile Self-Inhibitor
    Chitarra, G.S. ; Abee, T. ; Rombouts, F.M. ; Posthumus, M.A. ; Dijksterhuis, J. - \ 2004
    Applied and Environmental Microbiology 70 (2004)5. - ISSN 0099-2240 - p. 2823 - 2829.
    agaricus-bisporus - 10-oxo-trans-8-decenoic acid - colletotrichum-graminicola - pleurotus-ostreatus - spore germination - roqueforti - aspergillus - metabolism - mushroom - growth
    Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10(9) conidia ml(-1). By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25degreesC and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development.
    Endo-xylogalacturonan hydrolase
    Herweijer, M.A. ; Vincken, J.P. ; Meeuwsen, P.J.A. ; Vlugt-Bergmans, C.J.B. van der; Beldman, G. ; Ooyen, A.J.J. van; Voragen, A.G.J. - \ 2003
    In: Advances in pectin and pectinase research / Voragen, A.G.J., Schols, H.A., Visser, R.G.F., Dordrecht : - ISBN 9781402011443 - p. 257 - 266.
    pectinen - polygalacturonase - enzymen - appelsap - aspergillus - pectins - polygalacturonase - enzymes - apple juice - aspergillus
    A cDNA library of Aspergillus tubingensis was constructed in the yeast Kluyveromyces lactis, using a carbon source rich in xylogalacturonan. The library was screened using a hairy regions preparation from apple, and xylogalacturonan prepared from gum tragacanth as substrates. A novel endo-xylogalacturonase was found, XGH. The enzyme specifically degrades xylose-substituted galacturonic acid backbones. In lab scale filtration experiments, XGH was able to decrease membrane fouling caused by hairy regions from apple
    A molecular analysis of (hemi-)cellulose degradation by Aspergilli
    Gielkens, M.M.C. - \ 1999
    Agricultural University. Promotor(en): A.J.J. van Ooyen; L.H. de Graaff. - S.l. : S.n. - ISBN 9789058081384 - 136
    aspergillus - biodegradatie - cellulose - arabinose - aspergillus - biodegradation - cellulose - arabinose

    Glycosylhydrolases like cellulases and xylanases are of great importance for the ecological recycling of biomass. The saprophytic fungi, e.g Aspergillus niger , are capable of degrading plant cell wall material by secreting these enzymes. Because of their properties, a whole range of commercial enzyme preparations containing fungal polysaccharidases is used in industrial applications. For example, xylanases and cellulases are used in food and feed applications, and in pulp and paper applications, whereas cellulases are also used the textile industry in biostoning applications. The aim research described in this thesis is to clone genes encoding novel activities capable of degrading (hemi-)cellulose. Many of these novel activities are minor activities, which are difficult to detect and identify using standard conditions for growth. Several strategies will therefore be exploited to find these novel activities in Aspergilli. Firstly, the conditions of induction need to be optimal. This involves both the carbon source as well as the duration of growth (Chapter 4). Secondly, accumulation of the inducer often results in higher levels of expression (Chapter 3).

    Also the use of derepressed creAdstrains can result in elevated levels of expression as is shown in Chapters 2 and 3. Chapter 3 also shows that gene disruption of majorα-L-arabinofuranosidase can reveal minor activities which are otherwise difficult to identify. Finally, increasing the gene dosage of a specific transcriptional activator can increase the transcription levels of genes controlled by that activator (Chapters 5 and 6). Despite the fact that many glycosylhydrolases and their encoding genes have been isolated from a wide variety of microorganisms, little is known at the molecular level about the factors that are involved in the expression of these genes. The research described in this thesis gives a better understanding of the mechanisms underlying the regulation of expression of genes encoding cellulose- and hemicellulose-degrading enzymes produced by Aspergillus .

    Chapter 2 describes the isolation of A. niger creAdmutants relieved of carbon repression and the effects of the mutations on the expression of arabinanases and L-arabinose catabolic enzymes. Carbon repression is a global regulatory mechanism by which in the presence of D-glucose or other rapidly metabolisable carbon sources the expression of genes involved in the utilisation of less-favoured carbon sources is repressed. The arabinanolytic system was selected to illustrate that the expression of genes involved in the utilisation of less-favoured carbon sources could be enhanced in derepressed creAdmutant strains.

    Chapters 3 and 4 focus on genes encoding enzymes that are able to release L-arabinose from arabinoxylan. Chapter 3 describes the cloning and characterisation of the A. nidulans abfB gene, as well as the analysis of expression of the L-arabinofuranosidase encoding gene in an A. nidulans wild-type strain and several mutant strains. Super-induction of the abfB gene can be accomplished by combining a mutation leading to the intracellular accumulation of an inducer with a creAdmutation, as is shown by expression analyses. This chapter also describes the identification of minor L-arabinose releasing activities when the majorα-L-arabinofuranosidase activity was disrupted in a derepressed creAdgenetic background.

    The cloning, characterisation and analysis of expression of the axhA genes from the closely related fungi A. niger and A. tubingensis is described in Chapter 4. These genes encode an arabinoxylan-arabinofuranohydrolase A enzyme which specifically releases L-arabinose substituents from arabinoxylan. This chapter also describes the transcriptional analysis of the axhA and abfB genes in A. niger . It demonstrates that the regulation of transcription of the two genes differs significantly, although both genes encode L-arabinose releasing activities.

    The transcription of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in the degradation of cellulose in A. niger is studied in Chapter 5. In particular, the role of the xylanolytic transcriptional activator XlnR in the regulation of transcription of these genes was investigated. This analysis is extended in Chapter 6, which describes the cloning and characterisation of two cellobiohydrolase encoding genes in A. niger , cbhA and cbhB , which are also involved in the degradation of cellulose. The results described in Chapters 5 and 6 illustrate that the range of genes transcriptionally regulated by a specific activator is not necessarily limited to genes encoding pathway specific enzymes but that it also can include genes encoding non-pathway specific activities.

    Furthermore, the data described in these chapters give evidence that increasing the gene dosage of a specific transcriptional activator could elevate the expression of a broad range of genes controlled by that activator. Based on data described in this thesis a working model is proposed in which XlnR plays a central role in the degradation of plant cell walls.

    Horizontal transfer of genetic elements in the black Aspergilli
    Diepeningen, A.D. van - \ 1999
    Agricultural University. Promotor(en): R.F. Hoekstra; A.J.M. Debets. - S.l. : S.n. - ISBN 9789058080165 - 144
    gentransfer - aspergillus - gene transfer - aspergillus

    The thesis deals with the horizontal transfer of genetic elements in the black Aspergilli . The black Aspergilli form a complex group of asexual species. All share a characteristic black conidiospore color and the ability to efficiently degrade tannin. Selective isolation of all different black Aspergillus types is possible on media with 20% tannin. Tannins can form complexes with proteins, that are difficult to mineralize. Therefore, the strains may have a special niche in the control of the natural nitrogen cycle. Black Aspergilli occur worldwide and especially in warmer regions at high densities. The spores have an efficient aerial distribution, which produces a well-mixed sporebank in soil throughout the world.

    Under laboratory conditions isogenic lines are capable, after hyphal fusions, to form a heteroplasmic heterokaryon and (transient) diploids. This so-called parasexual cycle can result in recombination via reassortment of chromosomes, mitotic crossing-over and/or exchange of cytoplasmic genetic elements. Most of the natural isolates are heterokaryon incompatible with one another and unable to form a stable heterokaryon. About the exact mechanism of the heterokaryon incompatibility reactions in the black Aspergilli little is known. Confrontations between heterokaryon self-incompatible strains suggest that prefusion genes are involved. The fact that protoplast fusions are partly able to overcome incompatibility reactions suggests that also fusion and postfusion genes are involved.

    One of the cytoplasmic candidates for horizontal transfer is the mitochondrion. Different mitochondrial haplotypes can be distinguished, corresponding with different black Aspergillus types . No horizontal transfer or recombination of mitochondria was observed in our natural isolates, though in protoplast fusions mitochondria can recombine. In nature new mitochondrial types may result from mutations.

    Most of the transfer experiments in these thesis were done with cytoplamsic dsRNA mycoviruses. In nature 10% of the population is infected with a variety of different dsRNA fragments of different viral origins. These mycoviruses can cause serious reductions in their host's fitness on traits as spore production and growth rate. Population genetic models predict that deleterious elements should disappear from a population, unless they have an extra way of transfer than just vertical transmission to offspring. Interspecies transfer of mycoviruses with species like Fusarium poae was in our experiments less difficult to achieve (and thus perhaps more likely in nature) than intraspecies transfer between different black Aspergillus types.

    In a diploid both interchromosomal and intrachromosomal mitotic recombination could take place. However, molecular data suggests that there is little (para)sexual recombination in the black Aspergillus population, in contrast to other presumably asexual fungi tested so far. Recently transposable elements have been found in black Aspergillus strains. These do seem to have transposed between different, quite unrelated strains. Circular intermediates of these transposable elements may also transfer little parts of genomic DNA, which may lead to some recombination. The size of the genetic elements may influence the chance on horizontal transfer during cell contact: no detectable transfer of mitochondria, very little of mycoviruses and some transfer of transposable elements.

    Accessory enzymes from Aspergillus involved in xylan and pectin degradation
    Vries, R.P. de - \ 1999
    Agricultural University. Promotor(en): J.A.M. de Bont; J. Visser. - S.l. : De Vries - ISBN 9789058081087 - 192
    xylaan - pectinen - degradatie - aspergillus - enzymen - xylan - pectins - degradation - aspergillus - enzymes

    The xylanolytic and pectinolytic enzyme systems from Aspergillus have been the subject of study for many years. Although the main chain cleaving enzymes and their encoding genes have been studied in detail, little information is available about most of the accessory enzymes and their corresponding genes. This thesis describes the purification and characterisation of two accessory enzymes from Aspergillus , feruloyl esterase A (FaeA) andα-glucuronidase A (AguA), and the activities of these enzymes on polymeric substrates in relation to other accessory enzymes. Furthermore, the characterisation and regulation of the FaeA and AguA encoding genes ( fae A and agu A), and some genes encoding other accessory enzymes is studied.

    FaeA is the major feruloyl esterase produced when Aspergillus niger is grown on xylan or crude substrates such as wheat bran or sugar beet pulp. Addition of ferulic acid, the product of FaeA, to media containing xylan increases the production of this enzyme. FaeA is able to release ferulic acid from xylan and pectin oligosaccharides, as well as from synthetic substrates such as methylferulate. The fae A gene was cloned from A. niger and Aspergillus tubingensis . A blast of the deduced amino acid sequence of FaeA revealed no significant homology to other proteins, except for a small region of FaeA which was highly similar to the active site of lipases. Based on this homology, a 3-dimensional model for FaeA was proposed by Pickersgill et al. Although only 16 amino acid differences were observed between FaeA from A. niger and A. tubingensis , the latter enzyme was found to be much more sensitive to proteolytic degradation.

    AguA from A. tubingensis was able to release (4-O-methyl-) glucuronic acid from xylan derived oligosaccharides, but had very little activity towards the intact polysaccharide. The agu A gene was cloned, and database analysis of the deduced amino acid sequence revealed homology to theα-glucuronidases from Trichoderma reesei and Thermotoga maritima .

    Regulation of fae A and agu A expression was studied in A. niger and compared to other xylanolytic genes. Both genes were found to be under the control of the xylanolytic transcriptional activator protein XlnR, which also regulates endoxylanase,β-xylosidase, acetylxylan esterase, arabinoxylan arabinofuranohydrolase, and endoglucanase gene expression. In a XlnR negative mutant no expression of fae A and agu A was observed on xylose or xylan. Expression of fae A in this mutant was observed in the presence of ferulic acid, indicating the presence of a second system for the induction of this gene. This system seems to be specific for fae A, since no expression of agu A or other xylanolytic genes was observed under these conditions. In a wild-type A. niger strain, expression levels of fae A were higher on a combination of xylose and ferulic acid than the sum of the expression levels on xylose and ferulic acid alone, suggesting a synergistic effect of these two inducing systems.

    The carbon catabolite repressor protein CreA is involved in the repression of xylanolytic gene expression in the presence of easy metabolisable carbon sources, such as glucose or fructose. Expression of agu A and fae A on xylose and xylan, as well as expression of fae A on ferulic acid was repressed in the presence of glucose. Depending on the concentration of xylose present in the medium, this sugar also triggers CreA mediated repression of xylanolytic gene expression. Using a concentration range from 1 to 100 mM, it was shown that expression levels of fae A, agu A, and genes encoding endoxylanase B andβ-xylosidase decreased with increasing xylose concentrations in an A. niger wild type strain. In a CreA derepressed mutant constant levels of XlnR induced gene expression were observed indicating that the xylose concentration has a modulating effect via CreA.

    A gene ( agl B) encoding anα-galactosidase, which was produced when A. niger was grown on crude wheat arabinoxylan, was cloned and the expression of this gene was compared with the expression of two otherα-galactosidase encoding genes ( agl A and agl C) and aβ-galactosidase encoding gene ( lac A) from A. niger . All four genes had specific expression profiles with respect to monomeric sugars, galacto-oligosaccharides and polymeric substrates. High expression on xylan was only observed for agl B and lac A, suggesting that these genes may be part of the xylanolytic system from A. niger . This was confirmed using a XlnR negative mutant, which showed no ( lac A) or reduced ( agl B) expression of these genes on xylose.

    Synergy was studied between the accessory enzymes from Aspergillus involved in xylan degradation and two main chain cleaving enzymes, endoxylanase A (XlnA) andβ-xylosidase (XlnD). Except forα-L-arabinofuranosidase B (AbfB), the activity of all accessory enzymes on xylan was increased in the presence of XlnA. Similarly, the presence of accessory enzymes increased the activity of XlnA on xylan, indicating synergy between these enzymes. Synergy was also observed between the accessory enzymes, resulting in more efficient degradation of xylan. These results confirm that the gene products of XlnR regulated genes are in fact all part of the xylanolytic enzyme system of Aspergillus .

    Similarly, the synergy of enzymes involved in the degradation of the hairy regions in sugar beet pectin was studied. Degradation of the pectin backbone did not influence the activity of the arabinose releasing enzymes, AbfB and endoarabinase (AbnA), but had a strong effect on the release of ferulic acid by FaeA and the release of galactose by endogalactanase (GalA) andβ-galactosidase (LacA). Synergy was also observed between galactose- and ferulic acid- releasing enzymes.

    The accessory enzymes from Aspergillus involved in the degradation of xylan and pectin form a diverse group of enzymes which actively co-operate in polysaccharide degradation. Common factors have been identified in the regulation of the genes encoding these enzymes, but the expression patterns of the different genes also indicate the presence of other factors influencing specific genes. This most likely enables Aspergillus to modulate the production of these enzymes to obtain an efficient mixture for the degradation of the variety of substrates it encounters.

    On homologous recombination in Aspergillus nidulans
    Thijs, J.P. - \ 1998
    Agricultural University. Promotor(en): C. Heyting; T. Goosen; H.W.J. van den Broek. - S.l. : Thijs - 79
    recombinatie - crossing over - aspergillus - recombination - crossing over - aspergillus

    This thesis deals with the molecular mechanisms that underlie homologous recombination in eukaryotes. Most molecular genetic studies on homologous recombination have been performed in budding yeast ( Saccharomyces cerevisiae ). However, it remains to be investigated to what extent the results obtained in this unicellular organism can be extrapolated to other eukaryotes. We therefore initiated a series of molecular genetic studies on homologous recombination in Aspergillus nidulans . This filamentous fungus is amenable to most molecular techniques and is phylogenetically closer to higher eukaryotes than S. cerevisiae ; A. nidulans may thus provide a powerful eukaryotic model system. We have examined meiotic and mitotic recombination events in the nii A- nia D genomic region of A. nidulans . The nii A and nia D genes encode nitrite and nitrate reductase respectively, and are both required for the utilization of nitrate as a nitrogen source.

    The nii A- nia D region was chosen after a preliminary genetic analysis of meiotic recombination, which suggested the presence of an initiation site for meiotic recombination nearby, possibly in the intergenic promoter region (chapter 2). Subsequently, we designed an assay to analyze the products of recombination in more detail. As parental strains for a series of two-point crosses, we constructed nii A and nia D mutant strains that differed by nonsense or missense mutations in the nii A and/or nia D gene and by a series of 11 silent point mutations. We analyzed recombinants selected on nitrate for the presence or absence of individual mutations to determine the position and endpoints of conversion tracts.

    We found evidence for an initiation site for meiotic recombination in the promoter region located between the nii A and the nia D gene. Such initiation sites have also been found in the promoter regions of several genes of S. cerevisiae. Conversion tracts with an average length of 1.5 kb extended from this site into the nii A gene, the nia D gene or both. Meiotic conversion tracts were continuous in almost all recombinants. Co-conversion of markers declined steeply with distance from the proposed initiation site. In crosses where one of the parents harbored an ectopic copy of the nii A- nia D region instead of the allelic copy, conversion tracts were similar in size and position to those observed in allelic crosses. Ectopic recombinants were detected at a significantly lower frequency. The majority of the allelic recombinants but not of the ectopic recombinants harbored a crossover within the nii A- nia D region (chapter 3).

    Some initial molecular genetic studies on meiotic recombination in a higher plant (maize) have recently been published. In chapter 4, we draw attention to an important similarity between meiotic recombination in fungi and maize, namely a 5' to 3' polarity of gene conversion in the loci analyzed. It is thus possible that maize, like A. nidulans and S. cerevisiae , has specific initiation sites for meiotic recombination at the 5' ends of its genes.

    We also examined mitotic recombination in a diploid A. nidulans strain with the same assay system (chapter 5). The observed patterns of mutations in the haploid derivatives of the mitotic recombinants suggest that the initiation site for meiotic recombination that we found in the nii A- nia D locus, does not function in mitotic recombination; furthermore, the patterns suggested that long heteroduplex tracts (> 8.4 kb) are formed during mitotic recombination, and that conversion occurs in patches within these tracts. Mitotic recombination differs in these respects from meiotic recombination in A. nidulans .

    New rhamnogalacturonan degrading enzymes from Aspergillus aculeatus = Nieuwe rhamnogalacturonaan afbrekende enzymen uit Aspergillus aculeatus
    Mutter, M. - \ 1997
    Agricultural University. Promotor(en): A.G.J. Voragen; G. Beldman. - S.l. : Mutter - ISBN 9789054857822 - 141
    fermentatie - voedselbiotechnologie - groentesappen - vruchtensappen - aspergillus - fermentation - food biotechnology - vegetable juices - fruit juices - aspergillus

    Three new rhamnogalacturonan degrading enzymes were purified from a commercial enzyme preparation, Pectinex Ultra SP, produced by the fungus Aspergillus aculeatus . Pectinex Ultra SP is industrially used in the mash treatment of apples and pears in juice production, increasing juice yield. Rhamnogalacturonans are highly branched polysaccharides that are part of the pectin network in the plant cell wall. The purified enzymes were characterized and appeared to be only active toward rhamnogalacturonan and not toward the well-known pectic polysaccharide homogalacturonan. Rhamnogalacturonan rhamnohydrolase is able to remove the terminal nonreducing L-rhamnose residues which are α-(1,4)-linked to D-galacturonic acid residues in rhamnogalacturonans. Rhamnogalacturonan galacturonohydrolase is an enzyme able to remove the terminal nonreducing D-galacturonic acid residues which are α-(1,2)-linked to L-rhamnose residues in rhamnogalacturonans. Both enzymes were essential analytical tools in the study of the mode of action of rhamnogalacturonan hydrolase and a third new enzyme, rhamnogalacturonan lyase, toward linear rhamnogalacturonan oligosaccharides. While rhamnogalacturonan hydrolase cleaves α-D- galacturonic acid-(1,2)-L-rhamnose linkages by hydrolysis, rhamnogalacturonan lyase cleaves the α-L- rhamnose-(1,4)-D-galacturonic acid linkages by β-elimination. Both enzymes act in an endo-fashion, with a degree of multiple attack of 4 and 2.5 respectively toward modified hairy regions of apple. From the degree of multiple attack of these endo-enzymes, combined with information on the mode of action toward linear rhamnogalacturonan oligosaccharides, it could be estimated that the average length of rhamnogalacturonan regions In modified hairy regions of apple is at least 29 sugar residues.
    Molecular analysis of endo-rhamnogalacturonan hydrolases in Aspergillus
    Hoor - Suykerbuyk, M.E.G. ten - \ 1997
    Agricultural University. Promotor(en): N.C.M. Laane; J. Visser. - S.l. : Ten Hoor-Suykerbuyk - ISBN 9789054857068 - 145
    aspergillus - pectinen - enzymen - aspergillus - pectins - enzymes

    Bij de enzymatische bereiding van appelsap spelen pectinolytische enzymen en met nar-ne de enzymen die inwerken op de 'hairy regions' van pectine (MHR), een belangrijke rot. Schimmels zijn belangrijke enzym-producenten en een aantal Aspergillus soorten worden veelvuldig gebruikt in de voedingsmiddelenindustrie. In dit proefschrift staat de afbraak van MHR door rhamnogalacturonan hydrolases centraal en de produktie van deze enzymen is uitgebreid onderzocht in zowel A. aculeatus, de producent van het commerciële enzympreparaat Ultra SP, als A. niger.

    De waarneming van een endo-enzymactiviteit in Ultra SP, welke in staat is om MHR af te breken, betekende een doorbraak voor het inzicht in de wijze waarop pectine door schimmels wordt afgebroken. Tot dan toe waren alleen endo-enzymen bekend, die gericht zijn op de afbraak van homogalacturonan, zoals polygalacturonase, pectaat lyase en pectine lyase, en exo-enzymen, zoals galactosidase en arabinofuranosidase, die zich beperken tot de gedeeltelijke afbraak van MHR zijketens.

    Bij afbraak van MHR door rhamnogalacturonan hydrolase, worden tetra- en hexa- saccharide fragmenten uit de hoofdketen vrijgemaakt. Dit is niet geheel in overeenstemming met een alternerende volgorde van rhamnose en galacturonzuur in de hoofdketen en de herkenning van één van beide typen bindingen tussen deze monosacchariden door rhamnogalacturonan hydrolase. Blijkbaar vindt geen complete afbraak plaats en zijn voor verdere afbraak additionele endo-enzymen met andere specificiteiten nodig.

    In deze studie is allereerst het gen coderend voor rhamnogalacturonan hydrolase van A. aculeatus, rhg A, gecloneerd en het recombinant eiwit gezuiverd, zoals beschreven in hoofdstuk 2. Immunogene screening van een cDNA expressiebank resulteerde in een cDNA cloon, die vervolgens gebruikt werd als probe voor het screenen van een genomische bank.

    Kruishybridizatie van genomisch DNA van andere schimmelsoorten behorend tot de sectie Nigri met deze probe, resulteerde in meerdere hybridizerende DNA fragmenten en dus potentiële gen-families in alle stammen, maar vooral in A. niger. In eik van de onderzochte stammen werd tevens kruisreactiviteit met het polyclonale muize-antiserum, opgewekt tegen rhamnogalacturonan hydrolase van A. aculeatus, aangetoond. De betreffende eiwitten variëren in MW van 55 kDa in de verschillende onderzochte A. aculeatus en A. japonicus stammen tot 75 kDa in A. foetidus . De aanwezigheid van één rhamnogalacturonan hydrolase per stam leek niet in overeenstemming met de hybridizatie-patronen in de Southern analyse en tevens bleken deze enzymen algemeen binnen de sectie Nigri voor te komen.

    Het onderzoek naar rhamnogalacturonan hydrolase expressie werd daarom voortgezet in A, niger, een belangrijke producent van pectinolytische enzymen. In hoofdstuk 3 wordt de screening van het A. niger genoom naar sequenties homoloog aan het A. aculeatus rhgA gen beschreven. A. niger blijkt in het bezit te zijn van twee verwante genen, rhgA en rhgB. De hiervan afgeleide aminozuur sequenties vertonen ook, zij het in verschillende mate, overeenkomst met A. aculeatus rhamnogalacturonan hydrolase.

    Bij de vergelijking van de DNA sequenties bleken niet alleen de coderende gebieden, maar ook de aangrenzende, niet-coderende DNA sequenties homologieën te vertonen met het A. aculeatus gen. Beide rhg A promotoren hebben een box van 15 bp gemeenschappelijk en vertonen daarnaast 77% homologie over een traject van 130 bp. Mogelijk spelen deze elementen een regulerende rol bij de expressie van de verschillende rhamnogalacturonan hydrolase genen. De relatief geringe hoeveelheid rhamnogalacturonan in pectine in combinatie met de complexe structuur, maken het tot een moeilijk afbreekbaar substraat in vergelijking met bijv. homogalacturonan, dat vrij toegankelijk is voor enzymatische afbraak. Een verwachte opeenvolging in expressie van rhamnogaiacturonan afbrekende enzymen na homogalacturonan afbrekende enzymen blijkt niet te worden bevestigd. De expressie van rhg A in zowel A. aculeatus als A. niger is relatief vroeg. De genen komen in beide stammen reeds tot expressie binnen 6 h na transfer naar appel pectine. Expressie van het rhgB gen in A. niger kan alleen worden aangetoond op mRNA niveau na multicopy integratie in het genoom en is zichtbaar na 18 h groei op appel pectine maar ook op sucrose.

    Mogelijke verklaringen voor de lage expressie niveaus in A . niger zijn hoge proteolytische activiteit in de gebruikte stam en hoge gevoeligheid voor proteolytische afbraak van de betreffende rhamnogalacturonan hydrolases. De laatste verklaring is het meest waarschijnlijk, daar vergelijkbare resultaten werden verkregen na transformatie van A . aculeatus met deze genen.

    De toepassing van een enzym als rhamnogalacturonan hydrolase maakt een goed produktie systeem noodzakelijk. In de hoofdstukken 2 en 3 wordt een produktie systeem in A.awamori beschreven, dat gebaseerd is op de endogene endo-xylanase promoter. Dit maakt de produktie van rhamnogalacturonan hydrolases op xylose mogelijk i.p.v. op appel pectine, waardoor een zuiverder produkt verkregen wordt.

    Enzymactiviteiten van de A. aculeatus en A . niger rhamnogalacturonan hydrolases zijn vergeleken d.m.v. incubatie met MHR als substraat en analyse van de verkregen afbraakprodukten. Het A . niger B-enzym blijkt extra oligomeren te vormen waarvan een aantal met hoger MW, duidend op een andere substraat specificiteit dan de A-enzymen. Remming van het B-enzym door acetyl-groepen of andere zijketens is nog niet onderzocht. Echter, de aanwezigheid van extra monosacchariden, afkomstig van hoofdof zij-ketens van MHR, in sommige van de gevormde produkten, suggereert dat er verschillen in sterische hindering bestaan tussen het B-enzym en de A-enzymen. Mogelijk is het B-enzym minder afhankelijk van enzymen die bijv. de arabinogalactan zijketens afbreken.

    De specifieke activiteit van de verschillende rhamnogalacturonan hydrolases is afgeleid van de initiële snelheid waarmee één van de hexameren (corresponderend met piek Vlll in Fig. 7, hoofdstuk 3) gevormd wordt. Ondanks de relatief lage expressie-niveaus van rhamnogalacturonan hydrolase A en B in A. niger, vertonen deze enzymen een specifieke activiteit van respectievelijk 30% en 25% van die van A . aculeatus rhamnogalacturonan hydrolase. Vergelijking met gepubliceerde rhamnogalacturonan hydrolase-activiteiten, bepaald in andere organismen, wordt bemoeilijkt door het gebruik van verschillende detectie-methoden.

    De hoge mate van conservering van aminozuursequenties in de verschillende enzymen maakt toekenning van potentiële functies aan specifieke aminozuren onmogelijk en behoeft verdere analyse d.m.v. 'site-directed' mutagenese.

    In Aspergillus is nog niets bekend over mechanismen die de expressie van pectinolytische genen reguleren. De produktie van een breed scala aan MHR afbrekende enzymen in A. aculeatus na groei op pectine, duidt op een efficiënte pectine afbraak. Hiervoor is een goed-gecoördineerd regulatie-systeem, specifiek gericht op de afbraak van rhamnogalacturonan structuren, noodzakelijk. Kennis van de genstructuur van rhamnogalacturonan hydrolase maakt tal van moleculaire studies mogelijk. Een start ter identificatie van trans-factoren, die betrokken zijn bij de regulatie van deze genen, is gemaakt d.m.v. UV mutagenese van A. aculeatus. In hoofdstuk 4 wordt de isolatie van mutanten met verhoogde rhamnogalacturonan hydrolase expressie beschreven. De screening is uitgevoerd m.b.v. A . niger glucose oxidase als reporter systeem. Aangezien appel pectine een geschikte C-bron bleek voor de inductie van rhamnogalacturonan hydrolase in vloeibare kweken, werd dit substraat in eerste instantie ook gebruikt voor de screening van mutanten op agar medium. Echter de complexiteit van het substraat bracht een aantal nadelen met zich mee, zoals achtergrond kleuring, mogelijk represserende effecten door de aanwezigheid van andere suikers of inhibitie van de kleurreactie door interactie van pectine bestanddelen met H 2 0 2 . Een alternatieve C-bron voor de inductie van rhg A, gebaseerd op de combinatie van twee hoofdbestanddelen van rhamnogalacturonan, galacturonzuur en rhamnose, bleek wel bruikbaar. Mutanten met verschillende expressie patronen na groei op een aantal verschillende C-bronnen werden geselecteerd en vier van de mutaties konden d.m.v. complementatie-toetsen worden toegekend aan verschillende loci. Blijkbaar vindt regulatie van rhg A expressie in A. aculeatus plaats via een complex mechanisme, waarbij tenminste vier verschillende loci zijn betrokken. Identificatie van de betreffende trans-factoren zal bijdragen tot het ophelderen van het regulatie systeem en de rol van galacturonzuur en rhamnose daarin, maar ook mogelijkheden bieden tot een verdere verhoging van de enzymproduktie.

    Aangezien rhamnogalacturonan hydrolases worden gebruikt in de levensmiddelenindustrie, is het belangrijk dat de identiteit van een geselecteerde produktiestam gewaarborgd is. Dit vereist een gedegen classificatie methode. Gezien de arbitraire resultaten verkregen na classificatie van A . aculeatus stammen met de huidige methoden, welke veelal gebaseerd zijn op morfologische kenmerken, bestond de noodzaak tot een andere aanpak. In hoofdstuk 5 wordt een RFLP methode beschreven, waarmee A. aculeatus kan worden onderscheiden van een aantal andere soorten binnen de sectie Nigri, inclusief de morfologisch verwante A. japonicus. De groep van A. aculeatus stammen blijkt erg heterogeen te zijn vergeleken met bijv. de A . japonicus groep. Echter, een onderverdeling in variëteiten binnen beide groepen is op grond van deze classificatie niet langer gerechtvaardigd. Een aantal voormalige A. aculeatus en A . japonicus stammen wisselen van groep. Gezien de overeenkomstige morfologische kenmerken die beschreven zijn voor A. aculeatus en A. japonicus stammen, wordt een gemeenschappelijke phylogenetische voorouder, anders dan die van A. niger, waarschijnlijk geacht. Dezelfde RFLP methode is gebruikt voor het classificeren van A. carbonarius stammen en kan met geringe modificaties ook gebruikt worden voor de classificatie van andere Aspergilli.

    The transmission of cytoplasmic genes in Aspergillus nidulans
    Coenen, A. - \ 1997
    Agricultural University. Promotor(en): R.F. Hoekstra. - S.l. : Coenen - ISBN 9789054856542 - 106
    aspergillus - cytoplasmatische overerving - plasmiden - genen - genomen - eiwitsynthese - cytoplasma - aspergillus - cytoplasmic inheritance - plasmids - genes - genomes - protein synthesis - cytoplasm


    This manuscript concerns the spread of selfish cytoplasmic genes in the fungus Aspergillus nidulans. A.nidulans is a common soil fungus that grows vegetatively by forming a network (mycelium) of hyphae and reproduces via sexual ascospores and asexual conidiospores. Cytoplasmic genes are genes that are located in the cell cytoplasm and not in the cell nucleus where most genes are situated. The cytoplasmic genes investigated in this research are the genomes of mitochondria and viruses. Selfish genes are genes that are maintained in a population despite a negative effect on the fitness of their host.

    A possibility for the spread of selfish genes is created when they can compensate their negative effect on host fitness with an enhanced transmission rate. Because cytoplasmic genes can be transmitted independently from nuclear genes they can enhance their transmission rate in ways that nuclear genes cannot. Therefore there is evolutionary selection on mechanisms that regulate the transmission of cytoplasmic genes, thus preventing the spread of selfish cytoplasmic genes. Heterokaryon incompatibility may be such a mechanism. I have investigated the genetics of heterokaryon incompatibility and the transmission of mitochondria and viruses in A. nidulans. The implications of the results for the spread of selfish cytoplasmic genes are discussed.

    Genetics of heterokaryon incompatibility

    Contact between two fungal mycelia can result in their fusion and the formation of a heterokaryon (a mycelium containing nuclei from both strains). Such mycelial fusion creates possibilities for the horizontal transmission of cytoplasmic genes (transmission between individuals of the same generation). In most fungal species heterokaryon formation is restricted by a heterokaryon incompatibility mechanism. In A.nidulans heterokaryon incompatibility is regulated by heterokaryon incompatibility genes (het genes). Strains with different alleles for one or more het genes cannot form a heterokaryon, they belong to different heterokaryon compatibility groups (hcg's).
    Strains with the same alleles for all het genes can form a heterokaryon, they belong to the same hcg.

    A sample of 24 isolates collected in England and Wales in 1992 was found to contain 20 hcg's. Only 2 of these hcg's were present in the 20 previously described hcg's (Chapter 6). Due to this large amount of variation most pairwise strain combinations will be heterokaryon incompatible.

    Genetic analysis revealed the existence of partial heterokaryons, heterokaryons that grow less vigorously than 'normal' heterokaryons (Chapter 2). At first it was thought that partial heterokaryon incompatibility was caused by partial-het genes. Later it was discovered that partial heterokaryon incompatibility between parent and progeny could be induced by sexual reproduction (Chapter 3).

    I attempted to isolate strains in which heterokaryon incompatibility as a result of an allelic difference for het gene A or het gene B was suppressed (Chapter 3). 1 was successful for het gene B but not for het gene A. This may be because het gene A is an essential gene. All suppressor mutations were intragenic. Suppression of het gene B results in heterokaryon compatibility with both alleles of het gene B. Strains that had switched from compatibility with allele B' of het gene B to compatibility with allele B of het gene B were also isolated. This indicates that the two alleles are highly similar.

    Mitochondrial transmission

    Biparental inheritance creates possibilities for the spread of selfish mitochondrial genes. If mitochondria are inherited biparentally the progeny contain the mitochondria from both parents. Selfish mitochondria can enhance their transmission rate by winning the ensuing competition between the maternal and the paternal mitochondria. In A. nidulans mitochondrial inheritance is strictly uniparental (Chapter 5). All of the +-* 10000 ascospores in a fruitbody are inherited from the maternal parent. An investigation of more than a hundred fruitbodies did not reveal the presence of a single paternal mitochondrion. Selfish mitochondria cannot spread through A.nidulans populations by biparental inheritance.

    The horizontal transmission of mitochondria is greatly restricted. Horizontal transmission within a kg was observed in a very low frequency and horizontal transmission between hcg's was never observed (Chapter 6). Selfish mitochondria cannot spread through A. nidulans populations by horizontal transmission.

    The combination of uniparental inheritance and horizontal transmission within a kg creates a possibility for the spread of selfish mitochondria. The recombination of het genes during sexual reproduction results in the presence of the maternal mitochondria in all the hcg's included in the progeny. Consequently a selfish mitochondrion can spread by transmission within a hcg. However due to the low transmission rate within a hcg and the presumed rarity of sexual reproduction under natural conditions the spread of selfish mitochondria will be severely restricted. It is doubtful whether such a small enhancement of the mitochondrial transmission rate will compensate for the negative effects of selfish mitochondria, on host fitness.

    Virus Transmission

    There are no reports of viruses being found in sexual aspergilli despite their ubiquity in asexual aspergilli. For this research we transferred viruses from the asexual A..niger to the sexual A..nidulans by protoplast fusion (Chapter 4). Virus infection was not observed to have any effect on the fitness of Infected fungi. This shows that the absence of viruses from A..nidulans isolates is not the result of resistance to virus infection.

    The horizontal transmission of viruses within a hcg is highly efficient. The transmission between hcg's is restricted but not prevented by heterokaryon incompatibility. Heterokaryon incompatibility in itself will not prevent the spread of viruses through A. nidulans populations.

    Viruses are excluded from ascospores (Chapter 4). This will not prevent the spread of viruses through A. nidulans populations because viruses are included in the conidiospores. However the exclusion of viruses from ascospores and the recombination of het genes during sexual reproduction does allow the formation of new virus-free hcg's. If virus-free hcg's are created faster than they are infected by horizontal transmission this will result in the exclusion of viruses from A..nidulans populations. This could explain the exclusion of viruses from populations of sexual aspergilli.

    A molecular analysis of L-arabinan degradation in Aspergillus niger and Aspergillus nidulans
    Flipphi, M.J.A. - \ 1995
    Agricultural University. Promotor(en): A.J.J. van Ooyen; J. Visser. - S.l. : Flipphi - ISBN 9789054853923 - 165
    aspergillus - celwanden - koolhydraten - cellulose - celmembranen - fermentatie - voedselbiotechnologie - glycosidasen - polysacchariden - genexpressie - pleiotropie - moleculaire genetica - aspergillus - cell walls - carbohydrates - cellulose - cell membranes - fermentation - food biotechnology - glycosidases - polysaccharides - gene expression - pleiotropy - molecular genetics

    This thesis describes a molecular study of the genetics ofL-arabinan degradation in Aspergillus niger and Aspergillus nidulans. These saprophytic hyphal fungi produce an extracellular hydrolytic enzyme system to depolymerize the plant cell wall polysaccharideL-arabinan. Chapter 1 surveys the occurrence, properties and applications ofL-arabinanolytic enzymes (arabinases). The A.niger system, which constitutes an endolytic endo-1,5-α-L-arabinase (ABN A) and two distinct α-L-arabinofuranosidases (ABF A and ABF B), has been a frequent subject of investigation in the past and represents the best characterizedL-arabinanolytic system to date. These three enzymes are all glycosylated. Current knowledge on the induction of fungal arabinase expression is summarized in this Chapter. Furthermore, the structure of the polysaccharide substrate and its function in the plant cell wall matrix are introduced.

    In Chapters 2 to 5, the cloning and characterization of the structural genes coding for the three glycosyl hydrolases from the A. nigerL-arabinan-degrading complex are described. A. niger abf A and abf B ar e the first eukaryotic ABF-encoding genes to be isolated and sequenced, abn A is the first ABN-encoding gene published. Chapter 2 reports on the isolation of the abf A gene encoding ABF A, the minor extracellular ABF. This gene could be cloned by utilizing ABF Aspecific cDNA as the probe. This cDNA was immunochemically identified from a cDNA library generated fromL-arabitol-induced myceliurn of an A. nigerD-xylulose kinase mutant. This mutant is unable to grow onL-arabitol and features enhanced expression of all three arabinases when transferred to medium containing this pentitol as sole carbon source. In Chapter 3 , the cloning of the ABN A-encoding gene (abn A) is described. This gene was isolated following the same strategy as with abf A, although a second cDNA library had to be generated first. The induction process was immunochemically monitored in order to establish the proper induction conditions for the new library. The abn A gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and the cDN A. The N-terminal amino acid sequences of ABN A and a CNBr-derived peptide were determined. Several transcription initiation sites and one polyadenylation site could be identified. The structural region codes for a protein of 321 amino acids and is interrupted by three introns. Extracellular ABN A consists of 302 amino acid residues with a deduced molecular weight of 32.5 kDa and a theoretical pl of 3.5. For the protein, an apparent pl of 3.0 and an apparent molecular weight of 43 kDa, determined upon SDS-PAGE, were previously reported. Chapter 4 documents the isolation and characterization of the abf B gene, coding for the major extracellular ABF. The determination of N-terminal amino acid sequences from ABF B and CNBr-generated peptides allowed the design of deoxyoligonucleotide mixtures which enabled the cloning of abf B. When utilized as primers in a polymerase chain reaction (PCR), ABF B-specific amplification products emerged, one of which was used to probe the gene. The abf B gene and the gene product were characterized by DNA sequence analyses of the cloned genomic DNA and of ABF B- specific cDNA isolated from the library described in Chapter 3. Several transcription initiation sites and one polyadenylation site could be identified. The structural region is a single open reading frame and codes for a protein of 499 amino acids. The mature enzyme consists of 481 amino acid residues with a deduced molecular weight of 50.7 kDa and a theoretical pl of 3.8. An apparent pl of 3.5 and an apparent molecular weight of 67 kDa, determined upon SDS-PAGE, were previously reported. The abf B gene product was suggested to be identical to the ABF purified and characterized by Kaji and Tagawa (Biochim Biophys Acta 207 : 456-464 (1970)). Considering the non-amino acid content of the latter protein, a molecular weight of 64 kDa could be deduced for ABF B. In Chapter 5 , the abf A gene and its gene product were characterized by DNA sequence analyses of the genomic DNA and of the cDNA for which the isolation was described in Chapter 2. The N-terminal amino acid sequences of ABF A and a CNBr-derived peptide were determined. One transcription initiation site and two polyadenylation sites could be identified. The structural region is interrupted by seven introns and codes for a protein of 628 amino acids. Mature ABF A consists of 603 amino acid residues with a deduced molecular weight of 65.4 kDa and a theoretical pl of 3.7. For this ABF, an apparent pi of 3.3 and an apparent molecular weight of 83 kDa, determined upon SDS-PAGE, were previously documented.

    Although the three enzymes are all active against (1->5)-α-glycosidic bonds betweenL-arabinofuranosides, ABF A, ABF B and ABN A are genetically unrelated. ABF A was found to be N -glycosylated whereas ABF B and ABN A were not - these enzymes are only O -glycosylated. For each gene, arabinaseoverproducing strains were generated by introducing multiple gene copies in A.niger or in A.nidulans uridine auxotrophic strains through co-transformation. Transformants were isolated upon primary selection for uridine prototrophy. Subsequent overproduction of the genes introduced was demonstrated in these recombinant strains upon growth on sugar beet pulp, both immunochemically and by assaying enzyme activity. abf A was shown to be expressed in the heterologous host A.nidulans, despite the absence of an abf A gene equivalent in this organism. High-copy number A.niger abf B transformants featured impaired secretion of other extracellular proteins upon growth on sugar beet pulp. ABN A overproduction was found to be limited to approximately five times the wild-type level in A.niger abn A transformants, but not in A.nidulans transformants. Such a limitation was not observed in case of the ABFs.

    In Chapters 5 and 6, the regulation ofL-arabinan degradation is addressed. The structural genes seem to be regulated mainly at the transcriptional level. Additional copies of either A13F-encoding gene in A.niger were shown to result in a reduction, but not in total silencing of the expression of the wild-type ABN Aencoding gene upon induction with either sugar beet pulp orL-arabitol ( Chapter 5 ). The reduction of the expression level of abn A correlated with the abf gene dosage. The repression effected by extra abf B gene copies was more stringent and more persistent than that elicited by additional abf A copies. Although observed with both inducers, these phenomena were more outspoken and more persistent on sugar beet pulp. Similar, but more moderate effects were observed towards the expression of the other abf gene in multiple copy abf A- and abf B-transformants. It was proposed that the abf genes titrate two distinct gene activators both involved in coordination of arabinase gene expression. However, the three genes were shown to respond differently upon a mycelial transfer toL-arabitol-containing medium, indicating that gene-specific factors are also involved. Four distinct sequence motifs were found in common in the promoter regions of the three genes. One of these elements is identical to the A.nidulans CREA-motif, which has been shown to mediate carbon catabolite repression on several A.nidulans enzyme systems. Arabinase expression in A.niger is known to be repressed in the presence ofD-glucose. Two other motifs are highly homologous to cAMP-responsive elements described in other organisms. For the fourth motif no functional analogues could be found, but the element was found to be present in several other fungal genes which are not involved inL-arabinan degradation at all. It is therefore likely that none of these common elements confer system-specific regulation.

    The presumed involvement ofL-arabitol in the induction process of fungal arabinases was further emphasized by the induction characteristics of an A. nidulans mutant unable to grow on the end-product ofL-arabinan degradation,L-arabinose, nor onL-arabitol ( Chapter 6).L-Arabitol is an intermediate ofL-arabinose catabolism in Aspergilli. This mutant was shown to lack NAD +-dependentL-arabitol dehydrogenase activity resulting inL-arabitol accumulation, both intracellularly and in the culture medium, wheneverL-arabinose is present. Upon submerged growth on various carbon sources in the presence ofL-arabinose, the mutant featured enhanced expression of the enzymes involved in extracellularL-arabinan degradation, and of those of the intracellularL-arabinose catabolism. The co-substrates on which the mutant secreted large amounts of arabitol simultaneously exhibited high arabinase expression and featured reduced growth.L-Arabitol secretion and enzyme production were also observed on a mixed carbon source ofD-glucose andL-arabinose, resulting in normal growth. Hence, in the presence ofL- arabinose, the carbon catabolite repression conferred byD-glucose in the wild-type, is overruled in the mutant.

    In Chapter 7 , ABN A is shown to have remote sequence similarity with four bacterial xylanolytic glycosyl hydrolases (three β-D-xylosidases and an endo-1,4-β-D-xylanase), three of which feature activity against para -nitrophenyl-α-L-arabinofuranoside. This synthetic compound is commonly utilized to assay potential ABF activity, whereas it is known to be an inhibitor of the fourth enzyme. The homology became evident only after multi pie-sequence alignments and hydrophobic cluster analysis. It was proposed that these enzymes share a binding site for a terminal non-reducing α-linkedL-arabinofuranosyl residue and that they all belong to glycosyl hydrolase family 43. Implications from these suggestions were discussed. The ABFs could not be assigned to an established glycosyl hydrolase family.

    Based on theL-arabinolytic system of the brown-rot fungus Monilinia fructigena, the sequence similarity found amongst ABF A and bacterial pullulan-degrading enzymes, and ABF expression levels under carbon starvation conditions and onD-glucose as the carbon source, distinct functions inL-arabinan and plant cell-wall degradation were proposed for ABF A and ABF B. ABF A would be essentially cell-wall associated and act to degradeL-arabinan fragments generated by ABN A. ABF B activity would be important for the primary release of small amounts ofL-arabinose which initiate induction of various endolytic systems to degrade plant cell walls, and thus function in substrate sensing. In line with these considerations, the involvement of other, not yet identified glycosyl hydrolases inL-arabinan degradation by A.niger was suggested.

    Induction and repression of arabinase gene expression are further discussed in Chapter 7 . The results of the studies in A.niger (Chapter 5) and A.nidulans (Chapter 6) were interpreted in a mutual context. The identity of the lowmolecular-weight compound directly responsible for induction of arabinase gene expression, was addressed. BothL-arabinose andL-arabitol are likely candidates to fulfil such a role. However, it was not possible to weigh the actual inductive capacities ofL-arabinose andL-arabitol due to their in vivo convertibility and the carbon catabolite repression elicited by the pentose. Competition for such a compound provides an alternative explanation for the phenomena observed in Chapter 5. The involvement of the transcriptional repressor CREA in arabinase gene expression is not limited to the direct repression of structural and regulatory genes of theL-arabinan-degrading system. It also plays a role in inducer exclusion and end-product repression, two processes shown to be eminently involved in the regulation ofL-arabinan degradation in wild-type A.nidulans. Fungal growth rate was suggested to be related to derepression of theL-arabinan-degrading system. The possible involvement of cAMP in arabinase gene expression, as suggested by the presence of potential cis -acting cAMP-responsive elements in the structural genes, was considered. Various ways by which cAMP might modulate arabinase synthesis were surveyed.

    Arabinase induction and carbon catabolite repression in Aspergillus niger and Aspergillus nidulans
    Veen, P. van der - \ 1995
    Agricultural University. Promotor(en): A.G.J. Voragen; J. Visser. - S.l. : Van der Veen - ISBN 9789054853466 - 129
    aspergillus - hydrolasen - aspergillus - hydrolases

    The first aim of this thesis was to get a better understanding of the properties and the induction features of arabinan degrading enzymes and enzymes involved in the intracellular L-arabinose catabolic pathway in Aspergillus niger. The second aim was to understand the which role carbon catabolite repression plays in the induction process and to inventarize what phusiological consequences of an extreme carbon catabolite derepressed mutant are. The first part of this thesis the deals with the induction and characterisation of the arabinolytic enzyme system in Aspergillus niger. In the second part results of detailed studies on carbon catabolite repression of arabinase biosynthesis in Aspergillus nidulans are reported.

    In Chapter 2 several carbon sources were tested for their ability to induce arabinan degrading enzymes in A.niger N400. It was found that sugar beet pulp was the best inducing substrate for the induction of three arabinolytic enzymes. These three arabinan degrading enzymes were purified from the culture filtrate after growth on sugar beet pulp as the carbon source. The physico-chemical and kinetic properties of these three enzymes were characterised and found to correspond with those of the three arabinolytic enzymes purified from a commercial A . niger enzyme preparation by Rombouts et al. (1988). Also, the α-L-arabinofuranosidase B was found to correspond with an α-L-arabinofuranosidase B already characterised by Kaji and Tagawa (1970). Cloning and genetic analysis of the three genes by Flipphi et al (1993a; 1993b; 1993c) has shown that there are some discrepancies between the amino acid composition found by HPLC analysis and the one derived from the sequence data. This could be due to inaccuracies in HPLC analysis.

    Because of the finding in Chapter 2 that the monomeric sugar Larabinose induces arabinolytic activity, this sugar together with L-arabitol as an intermediate of the metabolic pathway of L-arabinose, was tested for inducing capacity in Chapter 3. In this case also an A.niger N572 xylulose kinase negative mutant strain was used which is disturbed in the last step of the metabolic pathway of L-arabinose, the conversion of D-xylulose to xylulose-5-phosphate (for a Scheme see Chapter 1 Fig. 2). This strain was characterised by Witteveen et al. (1989) by 13C- NMR analysis. Xylitol and L-arabitol were found to accumulate upon mycelial transfer to L-arabinose or D-xylose containing media.

    Using the sugars present in the metabolic pathway of L-arabinose and D-xylose it was found that within the wild type strain, L-arabinose induces α-L-arabinofuranosidase B but L-arabitol induces all three known arabinolytic enzymes. However, using the xylulose kinase negative mutant the level of induction on these two substrates was remarkable higher (up to 2 times). Moreover, in this mutant induction was also found using D-xylose and xylitol as a carbon source in transfer experiments. Of these two strains also the enzymes present in the catabolic pathway of L-arabinose were investigated. It was found in all cases that the activities in the xylulose kinase mutant were higher. The finding that L-arabitol dehydrogenase activity was almost 3 to 4 times higher on D-xylose and xylitol in comparison with the wild type was remarkable. Also induction of PNPA hydrolysing activity could already be detected using low amounts of L-arabitol (1.5 mM). Furthermore the induction of arabinases was found to be under the control of carbon catabolite repression. Induction on L-arabitol in the presence of 0.2% (w/v) D- glucose could only be detected when the concentration of D-glucose in the media became very low.

    These experiments lead to the conclusion that L-arabitol plays an important role in the induction of the arabinases in A. niger and that α-L-arabinofuranosidase B may trigger the biosynthesis of the other arabinases. It is known that the arabinolytic complex releases monomeric L-arabinose (Rombouts et al. 1988). This release of L-arabinose and its subsequent uptake by A. niger will convert the monomeric substrate into L-arabitol and thus induce all three arabinan degrading enzymes.

    Chapter 2 and 3 gave us information about the arabinan degrading enzyme system existing in A. niger and its induction, which could be activated by using simple low molecular weight substrates like L-arabitol an intracellular intermediate of the L-arabinose catabolic pathway. This induction system which involves both extra- and intracellular enzymes provides an excellent tool to study carbon catabolite repression of these enzymes in more detail. Chapter 4 and 5 describe experiments using Aspergillus nidulans, a fungus which has become a model organism for molecular genetic studies also because of the availability of a large collection of well defined mutant strains. In this case A. nidulans WGO96, which is used as the wild type strain, was first investigated for the presence of arabinolytic enzymes (Chapter 4). It was found that A. nidulans WG096 only produces 2 of the known arabinases, namely α-L-arabinofuranosidase B and endo-arabinase. α-L-arabinofuranosidase A could not be detected by Western blotting and also a genomic blot probed with the A. niger abf A gene did not give any positive signal. Biosynthesis of both arabinases was also found to depend on pathway-specific induction and carbon catabolite repression. Like in A. niger L-arabitol was also found to act as an efficient inducer for both arabinases present in A. nidulans.

    The observations made by De Vries et al. (1994) confirm the postulated role of L-arabitol in the induction mechanism of arabinan degrading enzymes. They have characterised an A . nidulans L-arabinose non-utilising mutant strain, which was first isolated by Roberts (1963) and characterised by Clutterbuck (1981) as an L- arabinose non-utilising mutant. It was found to be a L-arabitol dehydrogenase negative mutant strain. They also investigated the formation of polyols when transferred to L-arabinose containing media and found a strong accumulation of intracellular L-arabitol This accumulation of L-arabitol resulted in this strain in a strong induction of the two A.nidulans arabinases, which was confirmed both by enzymatic analysis as by Western blotting.

    The existence of some genetically well defined carbon catabolite derepressed mutants (Arst and MacDonald 1975; Arst and Bailey 1977; Dowzer and Kelly 1989; 1991) gave us an excellent tool to investigate the carbon catabolite repressing phenomena in more detail with respect to the induction of the two arabinases present in A. nidulans. In this case also the response of the intracellular L- arabinose degrading enzymes to carbon catabolite repression was investigated together with intracellular xylitol and L-arabitol measurements (Chapter 5). The data found indicate a strong influence of creA on the biosynthesis of both α-L- arabinofuranosidase B and endo-arabinase. The effect of the creA mutations was found to be most marked under inducing conditions. Both the more extreme carbon catabolite derepressed mutants cre A d-30 and cre A d-4 show a remarkable induction of arabinase activities up to 6- to 10 fold the level reached in the wild type suggesting a considerable 'self' repression in the wild type. In the presence of D-glucose plus an inducer the creA mutations, particularly cre A d-30, result in carbon catabolite derepression although the level of activity found did not reach the level which was found under inducing conditions. The intracellular enzymes present in the L- arabinose catabolic pathway show a more on-hierarchical behaviour. For example, whereas L-arabitol is a better inducer than L-arabinose of L-arabinose reductase in the cre A d-4 strain, the reverse is true for the cre A d-2 strain. Furthermore, from the finding that intracellular xylitol and thus L-arabitol could be detected on a mixed carbon source (D-glucose/L-arabinose) we can conclude that uptake of L-arabinose and conversion takes places even in the presence of D-glucose as a repressing carbon source.

    Although the direct role of the CREA protein in carbon catabolite repression by acting as a repressor of gene expression has now been elucidated (Kulmburg et al 1993; Cubero and Scazzocchio 1994), the physiological consequences of this mutation have not yet been investigated. Therefore a comparison was made on the level of enzyme activities and metabolite concentrations present in the metabolic pathway of D-glucose and polyol concentrations which can be derived from this route (Chapter 6). The enzymatic data obtained suggest that only a few enzymes are, directly or indirectly, influenced by the CREA protein. These are hexokinase and fructose-6-phosphate reductase, which were found to have elevated activities and phosphofructokinase and pyruvate kinase, which were found to show decreased activities within the cre A d-30 mutant. Both the metabolites fructose-2,6-diphosphate and fructose-1,6-diphosphate, which are the respective activators for the last two enzymes, were found to have increased internal concentrations. The result of these changes in activity is that this leads to a higher flux through the side chains towards the formation of polyols and possibly to a decreased flux through the TCA cycle due to the decreased activity of pyruvate kinase. The accumulation of polyols in the cre A d-30 mutant strain is remarkable. Although the amount of polyols produced intracellularly remains at a constant level in both strains, the total amount (intra- an extracellular) produced by the cre A d-30 mutant strain is almost up to times the total polyol concentration formed by the wild type strain However the nature of the repressing signal remains unclear.

    The induction of the arabinolytic enzyme system in A.nidulans was studied together with its response to carbon catabolite repression by using different carbon catabolite derepressed mutant strains. A study of the carbon catabolite repression on extracellular and intracellular enzymes together with an an easy induction system of these enzymes is scarce. The high induction of arabinase activity on L- arabitol in the extreme mutant strain cre A d-30 has almost the same level as found in wild type A . niger under normal induction conditions. In this case it would be interestingly which level of arabinase activity could be reached if carbon catabolite derepressed mutants of A. niger become available.

    Gluconate formation and polyol metabolism in Aspergillus niger
    Witteveen, C.F.B. - \ 1993
    Agricultural University. Promotor(en): W. Harder; J.A.M. de Bont; J. Visser. - S.l. : Witteveen - ISBN 9789054851387 - 135
    aspergillus - metabolisme - plantenvoeding - assimilatie - glucose - derivaten - polyethyleenglycol - glycolen - ethyleenglycol - alcoholen - thiolen - aspergillus - metabolism - plant nutrition - assimilation - glucose - derivatives - polyethylene glycol - glycols - ethylene glycol - alcohols - thiols

    The capacity of A.niger to accumulate metabolites is remarkable. Under all conditions polyols accumulate in the cell and when mycelium in later developmental stages is considered, depending on the carbon source, aeration and external pH, polyols and/or organic acids can be formed in a very efficient way. The aim of this thesis was to obtain a better understanding of the mechanisms governing the metabolism and formation of these metabolites. The first part of this thesis reports a study of gluconic acid formation and the second part involves polyol metabolism in A.niger.

    The fungus has a general tendency to synthesize organic acids under conditions of good aeration and when sugars like D-glucose or sucrose are available. An important function of the organic acid synthesis might be the acidification of the medium. Combined with the removal of the sugars from the environment this may contribute to the competitiveness of the fungus. A.niger is tolerant to pH values as low as 1.5. When glucose is the carbon source and the culture is well aerated A.niger will produce at neutral or slightly acidic pH mainly gluconate, at very low pH values citrate formation will occur and at intermediate pH oxalate is formed. The result is that A.niger by the consecutive synthesis of a series of organic acids strongly acidifies the medium.

    In this thesis the formation of gluconic acid was studied in more detail. In Chapter 2 evidence is provided for a cell wall localization of glucose oxidase. Furthermore, it was shown that two catalases are induced in parallel with glucose oxidase, one intracellular (CAT III) and one localized in the cell wall (CAT IV). Two other catalases, also one intracellular (CAT I) and one localized in the cell wall (CAT II), are constitutively present. About 50% of the lactonase activity was measured in the culture fluid. Therefore it was concluded that the whole glucose oxidase system is localized extracellularly, and is mainly localized in the cell wail. The induction of the intracellular CAT Ill may be seen as a second defense barrier, detoxifying hydrogen peroxide that diffuses into the cell.

    The cell wall-localization of glucose oxidase combined with the easy detection of hydrogen peroxide produced in the enzyme reaction makes visualization of the enzyme in intact hyphae possible. This detection system was used to isolate a series of mutants with altered glucose oxidase induction. In Chapter 3 a phenotypic and genetic characterisation of these mutants is presented. The mutants were classified in 9 different complementation groups, 1 non-producing, 1 low producing and 7 overproducing mutants. From induction experiments with the wild type strain it was concluded that the carbon source and the dissolved oxygen level are main factors determining induction of glucose oxidase. One mutant was found which never synthesized glucose oxidase ( gox C). Only one of the mutant classes was no longer dependent on oxygen for induction ( gox B). Several mutant groups were found with a decreased glucose dependency of induction. Some of these were quite strong ( gox A and gox E) whereas others showed only a minor overproduction under conditions which are only weakly inducing in the wild type. The latter group might in fact influence glucose oxidase induction only indirectly. The genetic analysis provided the information necessary for the construction of recombinant strains containing different gox mutations and other genetic markers. This is essential for further analysis and also an important step in further strain breeding.

    In Chapter 4 the induction mechanism of glucose oxidase, lactonase and the catalases was analyzed in more detail. For this we used beside the wild type strain gox B, gox C and gox E mutants. These mutants had a clear and pronounced phenotype. It was shown that in a wild type strain induction of all three activities is found only when glucose is present and the culture is well aerated. Induction of all three activities was effected by the gox B mutation. Neither glucose nor high oxygen levels were required for induction. The glucose dependency of glucose oxidase and lactonase induction was affected by the gox E mutation. In this mutant catalase was unaffected and high oxygen was still required. Thus with the gox B and gox E mutants the effects which oxygen and glucose have on induction could be partly separated. None of the activities was induced in the gox C mutant. This mutant could be transformed to a wild type phenotype using the structural gene of glucose oxidase, thus indicating that the mutation concerns the structural gene. The glucose oxidase structural gene was isolated via antibody screening of a cDNA expression library and subsequent homologous screening of a genomic library using the cDNA clone as a probe. Northern blotting showed that both the oxygen and carbon source effects were influencing induction at the transcriptional level. No glucose oxidase mRNA was observed in the gox C mutant. The absence of induction of all three activities in this mutant indicates that glucose oxidase activity is required for induction. It could be shown that hydrogen peroxide, besides gluconate a product of the glucose oxidase reaction, is inducing both catalase and lactonase in this mutant. It was concluded that hydrogen peroxide is the main factor required for the induction of the three activities and that the gox B gene product is involved in mediating this effect. It explained the requirement of high oxygen levels for induction because glucose oxidase has a high K m for oxygen (K m =0.48 mM at 27°C, Gibson etal., 1964). Even the glucose requirement for induction can be explained this way but the presence of the gox E mutant and the fact that the carbon source was still affecting the level of glucose oxidase in the gox B mutant, which is supposed to be involved in the transduction of the hydrogen peroxide signal, indicates that the induction process is more complicated. Using the plasmid plM503 which carries the structural gene for glucose oxidase multicopy transformants were made. Only a relatively small increase in glucose oxidase activity was observed (3 fold) even though more than 50 copies of the gene were integrated. Transformation of A.nidulans, a fungus that does not have the glucose oxidase gene itself, resulted in strains which produced glucose oxidase. In these strains glucose but not a high oxygen concentration was required for induction. This is indicating that a gox B-like gene is not present in A.nidulans but that the glucose requirement is presumably transduced by a more general system which is not specific for glucose oxidase.

    Chapters 2,3 and 4 are contributions to a better understanding of how the glucose oxidation system functions and of the molecular mechanism of its induction. Thus far it is the only coordinately regulated enzyme system in A.niger of which regulatory mutants have been isolated and which has been worked out in some detail. However, the understanding of the mechanism is still incomplete, especially the way by which the carbon source is affecting the induction is still unclear. No explanation is yet available why on fructose or D-xylose a basal level of glucose oxidase is found, whereas on acetate, gluconate or glycerol no activity is detected. Somehow the system senses the presence of an easy metabolisable carbon source and low-level induction occurs. The mechanism behind this phenomenon is probably not glucose oxidase-specific since in the A.nidulans transformants still glucose is required for induction. The gox B system is probably more glucose oxidase specific and therefore no oxygen (H 2 O 2 ) effect is found in A.nidulans. For a better understanding of the factors involved in glucose oxidase expression, a detailed analysis of the gox A and gox E mutants and of the functioning of the promoter of glucose oxidase is required. An important consequence of the regulatory mechanism hypothesized in Chapter 4 is a build-in feedback control. Catalase is not only induced by hydrogen peroxide but degrades this inducer as well, thus diminishing the induction. Ever increasing amounts of especially oxygen will be required to cause induction to continue. This will not happen because oxygen is quite soon the limiting factor in gluconate fermentation processes. The feedback mechanism of preventing overinduction is absent in gox B mutants. Therefore these mutants might be valuable in industrial processes.

    The function of the polyols is different from that of the organic acids. This is already clear from their presence in the fungus during all phases of the life cycle. Organic acids are formed only in late stages of development. Furthermore, the organic acids are excreted whereas the polyols beside being excreted also accumulate in large amounts in the mycelium. Information on carbon metabolism and more specifically polyol metabolism in A.niger was scarce at the start of this project. Therefore it was decided to analyze some metabolic pathways which directly relate to polyol metabolism.

    In Chapter 5 the characterization of a glycerol kinase mutant is described. Glycerol is one of the main polyols accumulating in A.niger and it was shown that glycerol kinase is involved in the degradation of glycerol. It could be demonstrated that the degradation pathway of glycerol in A.niger is largely the same as in A.nidulans (Hondmann et al., 1990) and N.crassa (Courtright 1975). First phosphorylation to glycerol-3-phosphate occurs and this is followed by its oxidation to dihydroxyacetonephosphate by a mitochondrial FAD-dependent glycerol-3- phosphate dehydrogenase. However, there were some differences with the pathway in A.nidulans. Whereas in the latter fungus dihydroxyacetone was catabolized via glycerol, in A.niger a dihydroxyacetone kinase was present enabling growth of the glycerol kinase mutant on dihydroxyacetone. Combined with an NAD +-dependent glycerol dehydrogenase converting glycerol into dihydroxyacetone this formed an escape route for glycerol catabolism in the glycerol kinase mutant. Growth on D-galacturonate was strongly affected in the glycerol kinase mutant thus demonstrating a D-galacturonate degradation pathway via glycerol.

    Pentose metabolism is described in Chapter 6. The isolation of a D-xylulose kinase mutant played an essential role in this work. It could be demonstrated that L-arabinose and D-xylose are catabolized via a series of reduction and oxidation steps. L-arabinose is reduced to L-arabitol which is oxidized to L-xylulose. L-xylulose is reduced to xylitol which is oxidized to D-xylulose that is phosphorylated to D-xylulose-5-phosphate, an intermediate of the pentose phosphate pathway. D-xylose is reduced to xylitol and subsequently oxidized to D-xylulose-5-phosphate. All the reduction steps are NADPH-dependent and all the oxidation steps NAD +-dependent The equilibrium of the reactions is far in the direction of the polyols, so several unfavourable steps that have to be taken which potentially can obstruct an efficient conversion of L-arabinose to D-xylulose-5- phosphate. The cofactor specificity of the dehydrogenases involved contributes to a higher efficiency since the anabolic reduction charge ([NADPH]/([NADPH]+[NADP +])) is higher than the catabolic reduction charge ([NADH]/([NADH]+[NAD +])) (Führer et al., 1980). A second mechanism for increasing the efficiency of this pathway was found by studying the two xylitol dehydrogenases of the L-arabinose pathway. This is described in Chapter 7. The NADPH-dependent L-xylulose reductase, catalyzing the reduction of L- xylulose to xylitol, was purified and the NAD +-dependent xylitol dehydrogenase, catalyzing the oxidation of xylitol to D-xylulose, was partially purified. Comparison of the two enzymes, which catalyze similar reactions leading to the different stereoisomers, made clear that they differ in two major points. 1) When their affinity for xylitol was compared it was found that the NAD +-dependent enzyme had a much higher affinity for xylitol than the NADPH-dependent enzyme. 2) Near the physiological pH (around 7) the ratio of the rductive relative to the oxidative catalytic activity was higher for the L-xylulose reductase than for the xylitol dehydrogenase. Both characteristics contribute to a more efficient catalysis of the reaction in the in vivo direction.

    In Chapter 8 an attempt is made to obtain some information on the function of the various polyol pools in A.niger. It was found that glycerol was the main polyol involved in osmotic adjustment in the fungus. Furthermore, glycerol accumulation was observed to be related to fast growing hyphae, whereas mannitol and erythritol accumulated in older hyphae. Mannitol also was an important storage compound in conidiospores. This general scheme of polyol accumulation during different growth phases is modified by environmental parameters like aeration of the culture and the nitrogen source available. Changes in fluxes through metabolic pathways and as a result of that changes in the steady state concentrations of intermediary metabolites from which the polyols derive, presumably play a role in this. It also implies that the function of the polyols in the cell is not completely coupled to specific polyols but can, in part, be taken over by other polyols. It was observed that a considerable part of the accumulated polyols (>50% after 24 h) is found in the medium. Observations made with the glycerol kinase mutant suggested that polyol excretion is a way for the fungus to control the intracellular levels of the polyols, presumably for maintaining the osmotic balance of the cell. Long fermentations (5 days) showed that in late developmental stages the polyol excretion becomes more pronounced. Approximately 45% of the glucose taken up was converted into extracelluar polyols. The type of the polyols excreted was a reflection of the intracellular polyol pool composition.

    Chapters 5 and 6 describe glycerol and pentose catabolism which has led to a better understanding of carbon metabolism in A.niger. Information on this subject is scarce in this fungus. The isolation of mutants in the degradation of such compounds is quite essential for metabolic studies. It turned out to be very difficult to isolate mutants in carbon metabolism in A.niger and still only a few of these mutants are available now. The reason for this is not known but the high intracellular polyol pools and the excretion of the polyols, resulting in crossfeeding during the filtration enrichment techniques, might play a role in this. The analysis of pentose metabolism and the xylitol dehydrogenases involved is also valuable in the light of understanding the mechanisms that play a role in extracellular enzyme production. The knowledge of the L-arabinose catabolic route has already proven useful in the analysis of the araban degrading system of A.niger. L-arabitol plays a major role in this (vd Veen et al., 1993). The analysis of polyol accumulation in Chapter 8 indicates that different polyols accumulate in different parts of the hyphae depending on their age. There still remains the question whether specific polyols accumulate in specific compartments of the cell, for example in the vacuoles, which are abundant in older hyphae. No information is available on this. It was shown that polyol excretion is a general phenomenon in A.niger. Although it has been observed before (Röhr et al., 1987), it was not considered to be such a common phenomenon in this fungus. The observation of large scale polyol accumulation in the culture fluid of 3-5 days old mycelial cultures grown under low oxygen conditions suggests similarities with organic acid fermentations. These are performed in strongly aerated cultures. An efficient flux to the TCA cycle is apparently not possible under the low aeration conditions used. This results in overflow metabolism in an earlier stage of the catabolic pathway leading to polyol formation. Cofactor regeneration might as well play a role in the polyol accumulation under these conditions.

    Extracellular polysaccharides as target compounds for the immunological detection of Aspergillus and Penicillium in food
    Kamphuis, H.J. - \ 1992
    Agricultural University. Promotor(en): F.M. Rombouts; S.H.W. Notermans. - S.l. : Kamphuis - ISBN 9789054850199 - 157
    voedselbesmetting - voedselmicrobiologie - aspergillus - penicillium - polysacchariden - food contamination - food microbiology - aspergillus - penicillium - polysaccharides

    This thesis is devoted to the immunological detection of Aspergillus and Penicillium in food products. More specifically, the immunogenicity, antigenicity, production and structure of the water-soluble extracellular polysaccharides (EPS) of these moulds have been studied, and a latex-agglutination assay, based on the detection of EPS has been developed.

    For the detection of moulds many methods are available, each of them with specific advantages and disadvantages, mostly related to reliability and applicability ( Chapter 2 ).

    An overview of the immunogenicity and antigenicity of EPS produced by moulds is presented in Chapter 3 . The role of β(1,5)-galactofuranoside sequences as epitopes of galactomannans from Aspergillus and Penicillium is documented. Antigenically specific polyclonal antibodies raised against P.digitatum EPS are directed towards β(1,5)-linked galactofuranosyl residues. These antibodies react specifically with EPS from Aspergillus and Penicillium.

    Synthetic tetramers and heptamers of β(1,5)-linked galactofuranosides are conjugated to tetanus toxoid and polyclonal antibodies are raised in rabbits against these synthetic immunogens ( Chapter 4 ). Antibodies obtained after immunisation with the heptamer conjugate possess the same genus specific antigenicity as the antibodies raised against P.digitatum EPS. No reactions are observed with the Penicillium subgenus biverticillium species and species belonging to genera other than Aspergillus and Penicillium . In contrast, antibodies raised against the tetramer conjugate reacted only with six out of 24 tested Aspergillus and Penicillium strains.

    From Glucanex, a Trichoderma harzianum enzyme preparation, an exo-β-D- galactofuranosidase is purified. This enzyme is used to hydrolyse specifically the immunodominant β(1,5)-linked galactofuranosyl residues from Aspergillus and Penicillium EPS. This enzyme alleviates the antigenicity of the EPS completely ( Chapter 5 ).

    Additionally, the reductive cleavage method for determination of the glycosidic bonds revealed that the β(1,5)-linked galactofuranosyl side chains in P.digitatum EPS carry side-chains of β(1,6)-1inked galactofuranosyl residues. These results allowed to propose a new structural model for the antigenically active galactofuranoside side chains of Penicillium galactomannans.

    In Chapter 6 , the production of antigenic EPS by P. aurantiogriseum and P. digitatum has been described under various growth conditions. Antigenic EPS was produced under almost all conditions investigated. However, both P.aurantiogriseum and P.digitatum do not produce antigenic EPS on lactate as the carbon source. Also, P.camemberti isolated from a mould fermented cheese (Camembert) does not produce antigenic EPS on lactate, althought, P.camemberti produces antigenic EPS on other substrates. The monosaccharide composition of the EPS produced by P.aurantiogriseum and P.digitatum under various conditions varies considerably.

    Immunopotent acid-labile β(1,5)-linked galactofuranosyl residues of Aspergillus fumigatus, Aspergillus niger and Penicillium digitatum EPS were acid-hydrolysed ( Chapter 7 ). Antibodies are raised against these acid-treated extracellular polysaccharides. It was supposed that these acid-treated EPS preparations would elicit antibodies with a broader specificity, making them useful in the detection of nearly all or all moulds occurring in food products. However, the antibodies obtained are more species specific and are generally directed to glucosyl and/or mannosyl residues of the EPS.

    Antibodies raised against P.digitatum EPS are used for the development of a rapid and reliable latex-agglutination assay for the detection of Aspergillus and Penicillium in food and feed ( Chapter 8 ). The reliability of the assay is enhanced by using a synthetic epitope, a tetramer of β(1,5)-Iinked galactofuranosides. With this tetramer false-positive results can easily be recognised.

    Finally, in Chapter 9 the applicability of the developed latex-agglutination assay is tested in both comparative and collaborative studies. The significance of extracellular polysaccharides produced by moulds for the detection of moulds in food and feed is discussed.

    Genetic analysis of Aspergillus niger
    Debets, F. - \ 1990
    Agricultural University. Promotor(en): R.F. Hoekstra; C.J. Bos. - S.l. : Debets - 104
    aspergillus - genetische modificatie - recombinant dna - genotypen - genetische variatie - aspergillus niger - aspergillus - genetic engineering - recombinant dna - genotypes - genetic variation - aspergillus niger

    Dit proefschrift handelt over genetische analyse van de voor de biotechnologie belangrijke schimmel Aspergillusniger . A.niger is een imperfecte schimmel, met andere woorden A.niger heeft geen geslachtelijk stadium, en mist daardoor meiotische recombinatie. Toch is genetisch onderzoek aan imperfecte schimmels mogelijk en wel op basis van af en toe optredende mitotische recombinatie in heterozygote diploiden. Twee typen recombinanten zijn hierbij van belang. Ten eerste kunnen door opeenvolgende non-disjunctie gebeurtenissen haploiden ontstaan die recombinant zijn als gevolg van hergroepering van chromosomen. Dergelijke recombinanten geven informatie omtrent de koppelingsgroep ('chromosoom') waartoe een bepaald gen behoort. Ten tweede kunnen door overkruisingen tussen homologe chromosomen diploide recombinanten ontstaan. Deze recombinanten kunnen worden gebruikt voor het in kaart brengen van genen die tot dezelfde koppelingsgroep behoren. De belangrijkste vereisten voor de genetische analyse van A.niger zijn: (1) De aanwezigheid van genetische markers. (2) Mogelijkheid tot selectie van de (zeldzame) recombinanten. (3) De mogelijkheid tot karakterisering van de recombinanten. Het primaire doel van het hier beschreven onderzoek was het ontwikkelen en toepassen van genetische technieken voor de constructie van een genetische kaart van A.niger . De genetische kaart kan vervolgens worden gebruikt voor genetisch onderzoek en bij de veredeling van stammen. De gevolgde strategie was tweeledig. Allereerst zijn methoden (verder) ontwikkeld waarmee genetische analyse op grond van de reeds aanwezige markergenen mogelijk is. Daarnaast zijn nieuwe markers geintroduceerd door mutatie en transformatie.

    In hoofdstuk 2 wordt een procedure beschreven voor de verrijking van auxotrofe recombinanten. De methode is gebaseerd op selectieve afdoding van geimmobiliseerde kiemende conidiosporen door celwand afbrekende enzymen (Novozym 234). De optimale condities voor deze 'Novozym-verrijking' zijn in simulatie experimenten bepaald. De methode is gebruikt voor het in kaart brengen van auxotrofe markers ten opzichte van het centromeer op chromosoom V.

    Hoofdstuk 3 beschrijft de isolatie en karakterisatie van chloraatresistente mutanten alsmede het gebruik ervan voor genetische analyse van A.niger . Deze resistentie mutaties behoren tot negen verschillende complementatiegroepen (plus één overlappende). De mutanten zijn onderverdeeld in drie klassen op grond van groei op verschillende stikstofbronnen. De chloraatresistentie genen liggen verspreid over zes koppelingsgroepen. Drie van deze markers bleken ongekoppeld met markers van de zes tot dan toe bekende koppelingsgroepen en leverden bewijs voor het bestaan van nog twee chromosomen in A.niger (n=8). De recessieve resistentie markers bleken zeer geschikt voor directe selectie van mitotische recombinanten. Ze zijn gebruikt voor het schatten van recombinatie-frequenties. Tevens is de lineaire volgorde van enkele markers op chromosoom VI bepaald aan de hand van direct selecteerbare overkruisingsprodukten. Enkele nitraatreductase mutaties zijn door transformatie met het A.nigernia D +gen gecomplementeerd. Van tien transformanten is de mitotische stabiliteit bepaald, en enkele zijn moleculair nader geanalyseerd.

    Hoofdstuk 4 beschrijft de genetische analyse van amd S transformanten van A.niger . Het A.nidulans amd S gen (coderend voor een aceetamidase) is door transformatie geintroduceerd in A.niger . De transformerende sequentie bleek in elk van de twaalf geanalyseerde transformanten in één koppelingsgroep geïntegreerd te zijn, waarschijnlijk in elke transformant op een unieke plaats. In totaal is op zeven van de acht chromosomen een amd S insertie gevonden. Onze (niet getransformeerde) A.niger stammen groeien niet op aceetamide en zijn minder gevoelig voor fluor-aceetamide dan de transformanten. Hierdoor is het mogelijk om naar twee kanten te selecteren: transformanten kunnen worden geselecteerd als groeiers op aceetamide, terwijl verlies van het AmdS +fenotype leidt tot fluoro-aceetamide resistentie. Diploiden die ontstaan zijn uit een transformant en een niet- transformant zijn hemizygoot voor de amd S insertie en zijn fenotypisch Amd +. De mitotische stabiliteit van het Amd +fenotype van transformanten en hemizygote diploiden is gekwantificeerd. De positie van het geïntegreerde plasmide ten opzichte van andere markers op het chromosoom bleek eenvoudig te bepalen.

    In hoofdstuk 5 is een genetische kaart van A.niger beschreven. Voor het bepalen van de ligging van 60 markers ten opzichte van het centromeer op de acht chromosomen is voornamelijk gebruik gemaakt van de genetische markers en technieken die in de hoofdstukken 2,3 en 4 beschreven zijn. Tevens is voor de genetische analyses gebruik gemaakt van amd S transformanten. Daartoe is de vermoedelijke positie van de amd S insertie van negen transformanten bepaald. In de meeste gevallen bleek de amd S marker distaal van de andere markers te liggen. Daardoor zijn de amd S transformanten zeer geschikt voor het in kaart brengen van (niet selecteerbare) recessieve markers omdat ze aan de volgende eisen voldoen:
    (1) Amd S transformanten zijn betrekkelijk eenvoudig te isoleren.
    (2) In hemizygote diploiden kan geselecteerd worden op recombinanten die de amd S marker kwijt zijn geraakt. De frequentie waarmee dergelijke recombinanten door overkruising ontstaan is afhankelijk van de relatieve afstand van de amd S marker tot het centromeer.
    (3) De analyse van recombinanten is eenvoudig: het genotype kan direct van het fenotype worden afgeleid.
    (4) De amd S marker kan op vele plaatsen in het genoom worden geïntegreerd, zodat in principe voor elke chromosoomarm een transformant met een distaal gelegen amd S marker kan worden verkregen.

    In hoofdstuk 6 is een electroforetisch karyotype van A. niger beschreven. Met behulp van 'pulsed-field' gel electroforese zijn de chromosomen van A.niger in vier banden gescheiden. Zeven van de acht koppelingsgroepen konden worden toegekend aan een specifieke chromosomale band. Hiervoor zijn zeven transformanten gebruikt die elk op een ander chromosoom de amd S marker dragen. De grootte van de chromosomen is geschat en varieert van 3,5 tot 6,6 megabasen. Electroforetische karyotypering is toegepast voor het idealiseren van A genen en voor het schatten van de grootte van de insertie bij een transformant met vele copien van het amd S plasmide.

    Hoofdstuk 7 tenslotte bevat een samenvatting en een algemene discussie.

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