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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    Commonalities and Differences in the Transcriptional Response of the Model Fungus Saccharomyces cerevisiae to Different Commercial Graphene Oxide Materials
    Laguna-Teno, Felix ; Suarez-Diez, Maria ; Tamayo-Ramos, Juan Antonio - \ 2020
    Frontiers in Microbiology 11 (2020). - ISSN 1664-302X
    biological response - chelating agent - commercial graphene oxide - differential expression - RNA isolation - Saccharomyces cerevisiae - transcriptomics

    Graphene oxide has become a very appealing nanomaterial during the last years for many different applications, but its possible impact in different biological systems remains unclear. Here, an assessment to understand the toxicity of different commercial graphene oxide nanomaterials on the unicellular fungal model organism Saccharomyces cerevisiae was performed. For this task, an RNA purification protocol was optimized to avoid the high nucleic acid absorption capacity of graphene oxide. The developed protocol is based on a sorbitol gradient separation process for the isolation of adequate ribonucleic acid levels (in concentration and purity) from yeast cultures exposed to the carbon derived nanomaterial. To pinpoint potential toxicity mechanisms and pathways, the transcriptome of S. cerevisiae exposed to 160 mg L–1 of monolayer graphene oxide (GO) and graphene oxide nanocolloids (GOC) was studied and compared. Both graphene oxide products induced expression changes in a common group of genes (104), many of them related to iron homeostasis, starvation and stress response, amino acid metabolism and formate catabolism. Also, a high number of genes were only differentially expressed in either GO (236) or GOC (1077) exposures, indicating that different commercial products can induce specific changes in the physiological state of the fungus

    Toxicological response of the model fungus Saccharomyces cerevisiae to different concentrations of commercial graphene nanoplatelets
    Suarez-Diez, Maria ; Porras, Santiago ; Laguna-Teno, Felix ; Schaap, Peter J. ; Tamayo-Ramos, Juan A. - \ 2020
    Scientific Reports 10 (2020)1. - ISSN 2045-2322

    Graphene nanomaterials have attracted a great interest during the last years for different applications, but their possible impact on different biological systems remains unclear. Here, an assessment to understand the toxicity of commercial polycarboxylate functionalized graphene nanoplatelets (GN) on the unicellular fungal model Saccharomyces cerevisiae was performed. While cell proliferation was not negatively affected even in the presence of 800 mg L−1 of the nanomaterial for 24 hours, oxidative stress was induced at a lower concentration (160 mg L−1), after short exposure periods (2 and 4 hours). No DNA damage was observed under a comet assay analysis under the studied conditions. In addition, to pinpoint the molecular mechanisms behind the early oxidative damage induced by GN and to identify possible toxicity pathways, the transcriptome of S. cerevisiae exposed to 160 and 800 mg L−1 of GN was studied. Both GN concentrations induced expression changes in a common group of genes (337), many of them related to the fungal response to reduce the nanoparticles toxicity and to maintain cell homeostasis. Also, a high number of genes were only differentially expressed in the GN800 condition (3254), indicating that high GN concentrations can induce severe changes in the physiological state of the yeast.

    Aspergillus niger citrate exporter revealed by comparison of two alternative citrate producing conditions
    Odoni, Dorett I. ; Vazquez-Vilar, Marta ; Gaal, Merlijn P. van; Schonewille, Tom ; Martins Dos Santos, Vitor A.P. ; Tamayo-Ramos, Juan Antonio ; Suarez-Diez, Maria ; Schaap, Peter J. - \ 2019
    FEMS Microbiology Letters 366 (2019)7. - ISSN 0378-1097
    Aspergillus niger - citrate - homology - MDR - transcriptomics - transport

    Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion. We hypothesise that depending on the experimental setup, extracellular citrate accumulation can have fundamentally different underlying transcriptomic landscapes. We show that varying the amount and type of supplement of an arginine auxotrophic A. niger strain results in transcriptional down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations when increased citrate production is triggered by iron limitation. By combining gene expression data obtained from these two very distinct experimental setups with hidden Markov models and transporter homology approaches, we were able to compile a shortlist of the most likely citrate transporter candidates. Two candidates (An17g01710 and An09g06720m.01) were heterologously expressed in the yeast Saccharomyces cerevisiae, and one of the resultant mutants showed the ability to secrete citrate. Our findings provide steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we demonstrate how a comparative transcriptomics approach complemented with further bioinformatics analyses can be used to pinpoint a fungal citrate exporter.

    Aspergillus niger N402 derivatives grown with different supplements
    Odoni, D.I. ; Vazquez Vilar, M. ; Gaal, Merlijn van; Schonewille, T. ; Martins dos Santos, V.A.P. ; Tamayo-Ramos, Juan Antonio ; Suarez Diez, M. ; Schaap, P.J. - \ 2018
    Wageningen University, Laboratory of systems and synthetic biology
    PRJEB24704 - PRJEB24704 - ERP106553 - Aspergillus niger
    Aspergillus niger N402 derivatives, harbouring an argB deletion and thus making them arginine auxothrophs, were grown with either 1.1 mM arginine or 5 mM citrulline added to the medium.
    A high quality Draft genome of Aspergillus niger N402 (ATCC 64974)
    Laothanachareon, T. ; Tamayo Ramos, J.A. ; Nijsse, B. ; Schaap, P.J. - \ 2018
    Wageningen University
    PRJEB21769 - PRJEB21769 - ERP024055 - Aspergillus niger
    A. niger strain N402 (cspA1) ATCC 64974) is a laboratory work horse strain that has been extensively used as a master strain for the generation of regulatory and structural mutants. Strain N402 is a derivative of the wild type strain N400 (CBS 120.49, NRRL3, ATCC 9029) and was obtained through two successive rounds of low-dosed UV mutagenesis.
    Forward Genetics by Genome Sequencing Uncovers the Central Role of the Aspergillus niger goxB Locus in Hydrogen Peroxide Induced Glucose Oxidase Expression
    Laothanachareon, Thanaporn ; Tamayo Ramos, J.A. ; Nijsse, Bart ; Schaap, Peter J. - \ 2018
    Frontiers in Microbiology 9 (2018)SEP. - ISSN 1664-302X - 14 p.
    Aspergillus niger is an industrially important source for gluconic acid and glucose oxidase (GOx), a secreted commercially important flavoprotein which catalyses the oxidation of β-D-glucose by molecular oxygen to D-glucolactone and hydrogen peroxide. Expression of goxC, the GOx encoding gene and the concomitant two step conversion of glucose to gluconic acid requires oxygen and the presence of significant amounts of glucose in the medium and is optimally induced at pH 5.5. The molecular mechanisms underlying regulation of goxC expression are, however, still enigmatic. Genetic studies aimed at understanding GOx induction have indicated the involvement of at least seven complementation groups, for none of which the molecular basis has been resolved. In this study, a mapping-by-sequencing forward genetics approach was used to uncover the molecular role of the goxB locus in goxC expression. Using the Illumina and PacBio sequencing platforms a hybrid high quality draft genome assembly of laboratory strain N402 was obtained and used as a reference for mapping of genomic reads obtained from the derivative NW103:goxB mutant strain. The goxB locus encodes a thioredoxin reductase. A deletion of the encoding gene in the N402 parent strain led to a high constitutive expression level of the GOx and the lactonase encoding genes required for the two-step conversion of glucose in gluconic acid and of the catR gene encoding catalase R. This high constitutive level of expression was observed to be irrespective of the carbon source and oxidative stress applied. A model clarifying the role of GoxB in the regulation of the expression of goxC involving hydrogen peroxide as second messenger is presented.
    Rhizopus delemar grown under high and low oxygen conditions
    Odoni, D.I. ; Tamayo Ramos, J.A. ; Sloothaak, J. ; Heck, R.G.A. van; Martins dos Santos, V.A.P. ; Graaff, L.H. de; Suarez Diez, M. ; Schaap, P.J. - \ 2017
    Wageningen University
    PRJEB14210 - PRJEB14210 - ERP015842 - Rhizopus delemar
    Rhizopus delemar grown under high and low oxygen conditions.
    Aspergillus niger N402 derivatives grown with different amounts of iron in the medium
    Odoni, D.I. ; Gaal, Merlijn van; Schonewille, T. ; Tamayo Ramos, J.A. ; Martins dos Santos, V.A.P. ; Suarez Diez, M. ; Schaap, P.J. - \ 2017
    PRJEB20746 - ERP022924 - Aspergillus niger - iron
    Aspergillus niger N402 derivatives were grown with either no iron added to the medium, or 10g/L Fe(II)SO4 added to the medium.
    Structure and function of Aspergillus niger laccase McoG
    Ferraroni, M. ; Westphal, A.H. ; Borsani, M. ; Tamayo Ramos, J.A. ; Briganti, F. ; Graaff, L.H. de; Berkel, W.J.H. van - \ 2017
    Biocatalysis 3 (2017)1. - ISSN 0886-4454 - p. 1 - 21.
    The ascomycete Aspergillus niger produces several multicopper oxidases, but their biocatalytic properties remain largely unknown. Elucidation of the crystal structure of A. niger laccase McoG at 1.7 Å resolution revealed that the C-terminal tail of this glycoprotein blocks the T3 solvent channel and that a peroxide ion bridges the two T3 copper atoms. Remarkably, McoG contains a histidine (His253) instead of the common aspartate or glutamate expected to be involved in catalytic proton transfer with phenolic compounds. The crystal structure of H253D at 1.5 Å resolution resembles the wild type structure. McoG and the H253D, H253A and H253N variants have similar activities with 2,2’-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid or N,N-dimethyl-p-phenylenediamine sulphate. However, the activities of H253A and H253N with 2-amino-4-methylphenol and 2-amino-4-methoxyphenol are strongly reduced compared to that of wild type. The redox potentials and electron transfer rates (ks) of wild type and variants were determined (McoG wt E°’ is +453 mV), and especially the reduced ks values of H253A and H253N show strong correlation with their low activity on phenolic compounds. In summary, our results suggest that the His253 adaptation of McoG can be beneficial for the conversion of phenolic compounds.
    Aspergillus niger secretes citrate to increase iron bioavailability
    Odoni, Dorett I. ; Gaal, Merlijn P. van; Schonewille, Tom ; Tamayo-Ramos, Juan A. ; Martins dos Santos, Vitor A.P. ; Suarez-Diez, Maria ; Schaap, Peter J. - \ 2017
    Frontiers in Microbiology 8 (2017)AUG. - ISSN 1664-302X
    Aspergillus niger - Citrate secretion - Iron homeostasis - Metabolic overflow - Siderophores
    Aspergillus niger has an innate ability to secrete various organic acids, including citrate. The conditions required for A. niger citrate overproduction are well described, but the physiological reasons underlying extracellular citrate accumulation are not yet fully understood. One of the less understood culture conditions is the requirement of growth-limiting iron concentrations. While this has been attributed to iron-dependent citrate metabolizing enzymes, this straightforward relationship does not always hold true. Here, we show that an increase in citrate secretion under iron limited conditions is a physiological response consistent with a role of citrate as A. niger iron siderophore. We found that A. niger citrate secretion increases with decreasing amounts of iron added to the culture medium and, in contrast to previous findings, this response is independent of the nitrogen source. Differential transcriptomics analyses of the two A. niger mutants NW305 (gluconate non-producer) and NW186 (gluconate and oxalate non-producer) revealed up-regulation of the citrate biosynthesis gene citA under iron limited conditions compared to iron replete conditions. In addition, we show that A. niger can utilize Fe(III) citrate as iron source. Finally, we discuss our findings in the general context of the pH-dependency of A. niger organic acid production, offering an explanation, besides competition, for why A. niger organic acid production is a sequential process influenced by the external pH of the culture medium.
    The uptake of carbon sources by Aspergillus niger
    Sloothaak, Jasper - \ 2017
    Wageningen University. Promotor(en): Vitor Martins dos Santos, co-promotor(en): P.J. Schaap; J.A. Tamayo-Ramos. - Wageningen : Wageningen University - ISBN 9789463432085 - 179
    aspergillus niger - carbon - proteomes - glucose - cell membranes - markov processes - organic acids - aspergillus niger - koolstof - proteomen - glucose - celmembranen - markov-processen - organische zuren

    Fungi have been used as food and in food fermentations long before written accounts were created and they have been used in folk medicine in ancient cultures. For centuries, species of the genus Aspergillus have been used for the preparation of traditional Asian foodstuffs or together with baker’s yeast in preparation of alcoholic beverages and have therefore been of great economical value. Later, Aspergillus niger has been used for large-scale production of organic acids, enzymes and other food-additives. Today, we aim to harness its saprophytic nature and extraordinary ability to degrade and utilize plant material that is naturally recalcitrant to degradation. To facilitate that ability, the range of sugar transporters employed by this fungus is large even among fungi. This makes it an excellent choice for the identification and characterization of a variety of proteins with different substrate specificities, with potential application in the design of newly engineered cell factories. This thesis was focused on the identification and characterization of previously unknown sugar uptake transporters. Aspergillus niger transporter proteins for the uptake of glucose, xylose, galacturonic acid and rhamnose were identified and characterized.

    Comparative proteomics of Rhizopus delemar ATCC 20344 unravels the role of amino acid catabolism in fumarate accumulation
    Odoni, Dorett I. ; Tamayo-Ramos, Juan A. ; Sloothaak, Jasper ; Heck, Ruben van; Martins dos Santos, Vitor A.P. ; Graaff, Leo H. de; Suarez-Diez, Maria ; Schaap, Peter J. - \ 2017
    PeerJ 5 (2017). - ISSN 2167-8359
    Amino acid metabolism - Fumarate - Nitrogen metabolism - Proteomics - Rhizopus delemar - Transcriptomics

    The filamentous fungus Rhizopus delemar naturally accumulates relatively high amounts of fumarate. Although the culture conditions that increase fumarate yields are well established, the network underlying the accumulation of fumarate is not yet fully understood. We set out to increase the knowledge about fumarate accumulation in R. delemar. To this end, we combined a transcriptomics and proteomics approach to identify key metabolic pathways involved in fumarate production in R. delemar, and propose that a substantial part of the fumarate accumulated in R. delemar during nitrogen starvation results from the urea cycle due to amino acid catabolism.

    Identification of a Novel L-rhamnose Uptake Transporter in the Filamentous Fungus Aspergillus niger
    Sloothaak, J. ; Odoni, D.I. ; Martins dos Santos, V.A.P. ; Schaap, P.J. ; Tamayo Ramos, J.A. - \ 2016
    Plos Genetics 12 (2016)12. - ISSN 1553-7404
    The study of plant biomass utilization by fungi is a research field of great interest due to its many implications in ecology, agriculture and biotechnology. Most of the efforts done to increase the understanding of the use of plant cell walls by fungi have been focused on the degradation of cellulose and hemicellulose, and transport and metabolism of their constituent monosaccharides. Pectin is another important constituent of plant cell walls, but has received less attention. In relation to the uptake of pectic building blocks, fungal transporters for the uptake of galacturonic acid recently have been reported in Aspergillus niger and Neurospora crassa. However, not a single L-rhamnose (6-deoxy-L-mannose) transporter has been identified yet in fungi or in other eukaryotic organisms. L-rhamnose is a deoxy-sugar present in plant cell wall pectic polysaccharides (mainly rhamnogalacturonan I and rhamnogalacturonan II), but is also found in diverse plant secondary metabolites (e.g. anthocyanins, flavonoids and triterpenoids), in the green seaweed sulfated polysaccharide ulvan, and in glycan structures from viruses and bacteria. Here, a comparative plasmalemma proteomic analysis was used to identify candidate L-rhamnose transporters in A. niger. Further analysis was focused on protein ID 1119135 (RhtA) (JGI A. niger ATCC 1015 genome database). RhtA was classified as a Family 7 Fucose: H+ Symporter (FHS) within the Major Facilitator Superfamily. Family 7 currently includes exclusively bacterial transporters able to use different sugars. Strong indications for its role in L-rhamnose transport were obtained by functional complementation of the Saccharomyces cerevisiae EBY.VW.4000 strain in growth studies with a range of potential substrates. Biochemical analysis using L-[3H(G)]-rhamnose confirmed that RhtA is a L-rhamnose transporter. The RhtA gene is located in tandem with a hypothetical alpha-L-rhamnosidase gene (rhaB). Transcriptional analysis of rhtA and rhaB confirmed that both genes have a coordinated expression, being strongly and specifically induced by L-rhamnose, and controlled by RhaR, a transcriptional regulator involved in the release and catabolism of the methyl-pentose. RhtA is the first eukaryotic L-rhamnose transporter identified and functionally validated to date.
    Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus Niger and Trichoderma reesei
    Sloothaak, Jasper ; Tamayo-Ramos, Juan Antonio ; Odoni, Dorett I. ; Laothanachareon, Thanaporn ; Derntl, Christian ; Mach-Aigner, Astrid R. ; Martins Dos Santos, Vitor A.P. ; Schaap, Peter J. - \ 2016
    Biotechnology for Biofuels 9 (2016)1. - ISSN 1754-6834
    Aspergillus Niger - Hidden Markov model - Str1 - Str2 - Str3 - Sugar porter - Transport kinetics - Trichoderma reesei - XltA - XltB - XltC - Xylose

    Background: Global climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus Niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes. Results: A hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. Niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. Niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. Niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization. Conclusions: This study revealed the existence of a variety of xylose transporters in the cell factories A. Niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. Niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. Niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.

    Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters
    Sloothaak, J. ; Odoni, D.I. ; Graaff, L.H. de; Martins dos Santos, V.A.P. ; Schaap, P.J. ; Tamayo Ramos, J.A. - \ 2015
    Biotechnology for Biofuels 8 (2015). - ISSN 1754-6834 - 15 p.
    BACKGROUND: The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic feedstocks into fermentable sugars. Both the hydrolytic enzymes responsible for lignocellulose depolymerisation and the molecular mechanisms controlling their expression have been well described, but little is known about the transport systems for sugar uptake in A. niger. Understanding the transportome of A. niger is essential to achieve further improvements at strain and process design level. Therefore, this study aims to identify and classify A. niger sugar transporters, using newly developed tools for in silico and in vivo analysis of its membrane-associated proteome. RESULTS: In the present research work, a hidden Markov model (HMM), that shows a good performance in the identification and segmentation of functionally validated glucose transporters, was constructed. The model (HMMgluT) was used to analyse the A. niger membrane-associated proteome response to high and low glucose concentrations at a low pH. By combining the abundance patterns of the proteins found in the A. niger plasmalemma proteome with their HMMgluT scores, two new putative high-affinity glucose transporters, denoted MstG and MstH, were identified. MstG and MstH were functionally validated and biochemically characterised by heterologous expression in a S. cerevisiae glucose transport null mutant. They were shown to be a high-affinity glucose transporter (K m = 0.5 ± 0.04 mM) and a very high-affinity glucose transporter (K m = 0.06 ± 0.005 mM), respectively. CONCLUSIONS: This study, focusing for the first time on the membrane-associated proteome of the industrially relevant organism A. niger, shows the global response of the transportome to the availability of different glucose concentrations. Analysis of the A. niger transportome with the newly developed HMMgluT showed to be an efficient approach for the identification and classification of new glucose transporters
    Enhanced glycosyl hydrolase production in Aspergillus nidulans using transcription factor engineering approaches
    Tamayo-Ramos, Juan Antonio ; Orejas, Margarita - \ 2014
    Biotechnology for Biofuels 7 (2014)1. - ISSN 1754-6834
    -L-rhamnosidase - Ambient pH regulation - Aspergillus nidulans - Endo-1,4-β-Xylanase - Fungal cell factories - PalA/PacC - Plant cell wall degrading enzymes - Protein production - Xylan/xylose regulator (XlnR)

    Background: Engineered fungi are attractive platforms for the production of plant cell wall hydrolytic enzymes which, among other biotechnological applications, are required for the efficient conversion of biomass to glucose and other fermentable sugars. As a fungal model system, Aspergillus nidulans provides genetic tools that are of relevance in this context and potentially applicable to industrially important filamentous fungi. The goal of this study is to assess the utility of A. nidulans as a host for recombinant protein production. Results: We have successfully applied a transcription factor engineering approach to improve the efficiency of the A. nidulans xylanolytic XlnR-xln p expression system. Specifically, endo-1,4-β-xylanases and an -L-rhamnosidase were chosen as representatives of endogenous and heterologous glycosyl hydrolases involved in plant cell wall deconstruction. By deregulating the expression of the xylanolytic transcriptional activator XlnR and modulating the activity of the pH regulator PacC we improved protein production and reduced production times. Xylanase activity was about 200-fold greater in gpdA p::xlnR strains compared to controls 4 hours after transfer to inducing conditions, and 10-fold greater after 24 hours. Remarkably, 75% of the xylanase activity was present in the engineered strains within 4 hours. Engineering XlnR expression also had a considerable impact on foreign protein production, especially when the promoter of the 'acidic' xlnB gene was used to express the transgene. -L-rhamnosidase activity in xlnB p::rhaA, gpdA p::xlnR strains was about 19-fold greater than that of controls 72 hours after transfer to xylan (about 85% of the total activity produced), and 10-fold greater at later times (120 hours). The performance of these strains was further enhanced by impeding the proteolytic activation of PacC; introduction of the palA1 allele in xlnB p::rhaA, gpdA p::xlnR strains resulted in an additional 2.7-fold increase in -L-rhamnosidase activity by 48 hours (about 87% of the total activity produced) and a 1.7-fold increase at later times. Conclusions: Our results show that the XlnR-xln p expression system is a valuable tool for manipulating the production of plant cell wall degrading enzymes in A. nidulans and establish the biotechnological potential of the transcription factors XlnR and PacC to boost and control the strength of xylanolytic promoters.

    Fungals strains with improved citric acid and itaconic acid production
    Straat, L. van der; Schonewille, T. ; Tamayo Ramos, J.A. ; Graaff, L.H. de - \ 2014
    Octrooinummer: WO2014142647, gepubliceerd: 2014-09-18.
    The present invention relates to cells with improved citric acid and itaconic acid production and to the use of these cells for the production of citric acid and itaconic acid.
    Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans
    Heshof, R. ; Schayck, J.P. van; Tamayo Ramos, J.A. ; Graaff, L.H. de - \ 2014
    AMB Express 4 (2014). - ISSN 2191-0855 - 6 p.
    niger - protein - biosynthesis - oxylipins - genes
    Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.
    Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger
    Straat, L. van der; Tamayo Ramos, J.A. ; Schonewille, T. ; Graaff, L.H. de - \ 2013
    AMB Express 3 (2013). - ISSN 2191-0855
    A modified 6-phosphofructo-1-kinase was expressed in a citrate producing Aspergillus niger strain in combination with cis-aconitate decarboxylase from Aspergillus terreus to study the effect on the production of itaconic acid. The modified pfkA gene was also expressed in combination with the itaconic acid biosynthetic cluster from A. terreus, which consists of cis-aconitate decarboxylase cadA, a putative mitochondrial transporter mttA and a putative plasmamembrane transporter mfsA. The combined expression of pfkA and cadA resulted in increased citrate levels, but did not show increased itaconic acid levels. The combined expression of pfkA with the itaconic acid biosynthetic cluster resulted in significantly increased itaconic acid production at earlier time points. Also the itaconic acid productivity increased significantly. The maximum itaconic acid productivity that was reached under these conditions was 0.15 g/L/h, which is only a factor 17 lower than the 2.5 g/L/h that according to the US Department of Energy should be achieved to have an economically feasible production process
    Enhanced production of Aspergillus niger laccase-like multicopper oxidases through mRNA optimization of the glucoamylase expression system
    Tamayo Ramos, J.A. ; Barends, S. ; Lange, D. ; Jel, A. de; Verhaert, R.M. ; Graaff, L.H. de - \ 2013
    Biotechnology and Bioengineering 110 (2013)2. - ISSN 0006-3592 - p. 543 - 551.
    5'-untranslated region - protein expression - gene - translation - transformation - promoter - nidulans - cloning - oryzae
    In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5' untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5'UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5'UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5'UTR can lead to improvements in protein yields. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc
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