Browse by Lectorate "Biodiscovery"

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The role of Coproporphyrinogen III oxidase and Ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger
Hondel, C.A. van den ; Punt, P.J. ; Haas, H. ; Ram, Arthur F.J. ; Werner, M. ; Weert, S. de ; Franken, A.C. ; Lokman, B.C.
2013, Article / Letter to editor (Applied Microbiology and Biotechnology, vol. 97, iss. 22, (2013), pp. 9773-9785)Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.
Selection of oleaginous yeasts for fatty acid production
Lamers, W.L. ; Biezen, N. van ; Martens, D. ; Peters, L. ; Zilver, E. van de ; Jacobs-van Dreumel, N. ; Wijffels, R.H. ; Lokman, B.C.
2016, Article / Letter to editor (vol. 16, iss. 1, (2016), pp. 45)Background Oleaginous yeast species are an alternative for the production of lipids or triacylglycerides (TAGs). These yeasts are usually non-pathogenic and able to store TAGs ranging from 20 % to 70 % of their cell mass depending on culture conditions. TAGs originating from oleaginous yeasts can be used as the so-called second generation biofuels, which are based on non-food competing “waste carbon sources”. Results In this study the selection of potentially new interesting oleaginous yeast strains is described. Important selection criteria were: a broad maximum temperature and pH range for growth (robustness of the strain), a broad spectrum of carbon sources that can be metabolized (preferably including C-5 sugars), a high total fatty acid content in combination with a low glycogen content and genetic accessibility. Conclusions Based on these selection criteria, among 24 screened species, Schwanniomyces occidentalis (Debaromyces occidentalis) CBS2864 was selected as a promising strain for the production of high amounts of lipids.
Prominent Inhibitory Projections Guide Sensorimotor Computation: An Invertebrate Perspective
Hughes, S.L. ; Celikel, T.
2019, Article / Letter to editor (vol. 41, iss. 10, (2019))From single-cell organisms to complex neural networks, all evolved to provide control solutions to generate context- and goal-specific actions. Neural circuits performing sensorimotor computation to drive navigation employ inhibitory control as a gating mechanism as they hierarchically transform (multi)sensory information into motor actions. Here, the focus is on this literature to critically discuss the proposition that prominent inhibitory projections form sensorimotor circuits. After reviewing the neural circuits of navigation across various invertebrate species, it is argued that with increased neural circuit complexity and the emergence of parallel computations, inhibitory circuits acquire new functions. The contribution of inhibitory neurotransmission for navigation goes beyond shaping the communication that drives motor neurons, and instead includes encoding of emergent sensorimotor representations. A mechanistic understanding of the neural circuits performing sensorimotor computations in invertebrates will unravel the minimum circuit requirements driving adaptive navigation.
Production of tailor-made enzymes to facilitate lipid extraction from the oleaginous yeast Schwanniomyces occidentalis
Heshof, R. ; Visscher, B. ; Zilver, E. van de ; Vondervoort, R. van de ; Keulen, F. van ; Delahaije, R.J. ; Wind, R.D.
2020, Article / Letter to editor (vol. 10, iss. 1, (2020), pp. 1-11)Due to the depletion of fossil fuel resources and concern about increasing atmospheric CO2 levels, the production of microbial oil as source for energy and chemicals is considered as a sustainable alternative. A promising candidate strain for the production of microbial oil is the oleaginous yeast Schwanniomyces occidentalis CBS 2864. To compete with fossil resources, cultivation and processing of S. occidentalis requires improvement. Currently, different cell wall disruption techniques based on mechanical, chemical, physiological, and biological methods are being investigated using a variety of oil producing yeasts and microalgae. Most of these techniques are not suitable for upscaling because they are technically or energetically unfavorable. Therefore, new techniques have to be developed to overcome this challenge. Here, we demonstrate an effective mild enzymatic approach for cell disruption to facilitate lipid extraction from the oleaginous yeast S. occidentalis. Most oil was released by applying 187 mg L−1 tailor-made enzymes from Trichoderma harzianum CBS 146429 against the yeast cell wall of S. occidentalis at pH 5.0 and 40 °C with 4 h of incubation time after applying 1 M NaOH as a pretreatment step.
Overexpression of delta-12 desaturase in the yeast Schwanniomyces occidentalis enhances the production of linoleic acid.
Lamers, W.L. ; Visscher, B. ; Weusthuis, R.A. ; Francke, C. ; Wijffels, R.H. ; Lokman, B.C.
2019, Article / Letter to editor (Bioresource Technology, vol. 289, (2019), pp. 121672)The oleaginous yeast Schwanniomyces occidentalis was previously isolated because of its excellent suitability to convert lignocellulosic hydrolysates into triacyl glycerides: it is able to use a broad range of sugars and is able to tolerate high concentrations of lignocellulosic hydrolysate inhibitors. Compared to other oleaginous yeasts S. occidentalis however produces a low content of unsaturated fatty acids. We show here that the linoleic acid content can be significantly improved by (over)expression Δ12-desaturases derived from S. occidentalis and Fusarium moniliforme. Expression was stable for the homologous expression but decreased during heterologous expression. Both homologous and heterologous expression of mCherry-Δ12-desaturase led to a 4-fold increase in linoleic acid from 0.02 g/g biomass to 0.08 g/g biomass resulting in the production of 2.23 g/L and 2.05 g/L of linoleic acid.
A Novel Quality Measure and Correction Procedure for the Annotation of Microbial Translation Initiation Sites.
Overmars, L. ; Siezen, R.J. ; Francke, C.
2015, Article / Letter to editor (PLoS One, vol. 10, iss. 7, (2015), pp. e0133691)The identification of translation initiation sites (TISs) constitutes an important aspect of sequence-based genome analysis. An erroneous TIS annotation can impair the identification of regulatory elements and N-terminal signal peptides, and also may flaw the determination of descent, for any particular gene. We have formulated a reference-free method to score the TIS annotation quality. The method is based on a comparison of the observed and expected distribution of all TISs in a particular genome given prior gene-calling. We have assessed the TIS annotations for all available NCBI RefSeq microbial genomes and found that approximately 87% is of appropriate quality, whereas 13% needs substantial improvement. We have analyzed a number of factors that could affect TIS annotation quality such as GC-content, taxonomy, the fraction of genes with a Shine-Dalgarno sequence and the year of publication. The analysis showed that only the first factor has a clear effect. We have then formulated a straightforward Principle Component Analysis-based TIS identification strategy to self-organize and score potential TISs. The strategy is independent of reference data and a priori calculations. A representative set of 277 genomes was subjected to the analysis and we found a clear increase in TIS annotation quality for the genomes with a low quality score. The PCA-based annotation was also compared with annotation with the current tool of reference, Prodigal. The comparison for the model genome of Escherichia coli K12 showed that both methods supplement each other and that prediction agreement can be used as an indicator of a correct TIS annotation. Importantly, the data suggest that the addition of a PCA-based strategy to a Prodigal prediction can be used to 'flag' TIS annotations for re-evaluation and in addition can be used to evaluate a given annotation in case a Prodigal annotation is lacking.
Montmorency Tart Cherry (Prunus cerasus L.) acts as a calorie restriction mimetic that increases intestinal fat and lifespan in Caenorhabditis elegans
Klashorst, D. van de ; Elzen, A. ; Weeteling, J. ; Roberts, M. ; Desai, T. ; Bottoms, L. ; Hughes, S.L.
2020, Article / Letter to editor (vol. 68, iss. May 2020, (2020))Montmorency Tart Cherries, MTC, (Prunus cerasus L.) possess a high anthocyanin content as well as one of the highest oxygen radical absorbance capacities of fruits at common habitual portion sizes. MTC have been shown to contribute to reducing plasma lipids, plasma glucose and fat mass in rats and strikingly, similar effects are observed in humans. However, there is a paucity of research examining the molecular mechanisms by which such MTC effects are induced. Here, we show that when exposed to MTC, Caenorhabditis elegans display an extension of lifespan, with a corresponding increase in fat content and increase in neuromuscular function. Using RNA interference, we have confirmed that MTC is likely to function via the Peroxisome Proliferator-Activated Receptor (PPAR) signalling pathway. Further, consumption of MTC alters the pharyngeal pumping rate of worms which provides encouraging evidence that MTC may be operating as a calorie restriction mimetic via metabolic pathways.
Leren voor morgen : Trends in onderwijs en de implicaties daarvan voor de lerarenopleidingen
Eldik, Sabine van ; Lokman, B.C. ; Vijlder, F.J. de ; Bakker, Dorine ; Peters, M.J. ; Kingma, Marius
2011, External research report De lerarenopleidingen vervullen een scharnierfunctie in ons onderwijsbestel. In de lerarenopleidingen worden jonge mensen voorbereid op de vervulling van een belangrijke publieke taak, namelijk het verzorgen van onderwijs aan leerlingen in het primair en voortgezet onderwijs en in het middelbaar beroepsonderwijs. ‘Leren voor morgen’ betekent daarom dat de lerarenopleidingen een spilfunctie moeten vervullen in de innovatie van het onderwijs, moeten voorbereiden op zich ontluikende innovaties in dat onderwijs én actie moeten bijdragen aan die innovaties door middel van onderzoek. Wie leraar is, draagt een bijzondere maatschappelijke verantwoordelijkheid. Dus wat betekent dat voor degenen die leraren opleiden en professionaliseren?
Large-scale cultivation of Caenorhabditis elegans in a bioreactor using a labor-friendly fed-batch approach.
Heshof, R. ; Visscher, B. ; Prömel, S. ; Hughes, S.L.
2019, Article / Letter to editor (vol. 67, iss. 1, (2019), pp. 33-39)Caenorhabditis elegans is an invertebrate model organism used in many areas of biology including developmental biology and the identification of molecular mechanisms and pathways. However, several experimental approaches require large quantities of worms, which is limiting and time-consuming. We present a protocol that uses modern fermentation methodology to effectively produce large numbers of C. elegans using a 7-l bioreactor in a fed-batch cultivation procedure. The production is modular and flexible as well as being a self-controlled system, thus not much labor is required until harvesting C. elegans. The high-yield worm cultivation is flexible and simple to amend, and now allows for the extended application of C. elegans as a model organism and expression system, including large-scale protein production.
Heterologous Expression of Peroxidases : Chapter 12
Lokman, B.C. ; Weert, S. de
2010, Part of book or chapter of book ()This monograph describes many applications of peroxidase-based biocatalysis in the biotechnology industry. The need for such a book emerges from the considerable amount of new data regarding the phylogeny, reaction mechanisms, thermodynamic characterization and structural features of fungal and plant heme peroxidases that has been generated in the past 10 years, since the last specialized book on peroxidases was published. The aim of this book is to present recent advances on such basic aspects as evolution, structure-function relation and catalytic mechanism as well as applied aspects, such as bioreactor and protein engineering, to provide the tools for rational design of enhanced biocatalysts and biocatalytic processes.
Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.
Weert, S. de ; Punt, P.J. ; Lokman, B.C. ; Hondel, C.A. van den ; Franken, A.C. ; Ram, Arthur F.J. ; Ram, A.F.
2011, Article / Letter to editor (Applied Microbiology and Biotechnology, vol. 91, iss. 3, (2011), pp. 447-460)Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally produced in small amounts by basidiomycetes. Filamentous fungi like Aspergillus sp. are considered as suitable hosts for protein production due to their high capacity of protein secretion. For the purpose of peroxidase production, heme is considered a putative limiting factor. However, heme addition is not appropriate in large-scale production processes due to its high hydrophobicity and cost price. The preferred situation in order to overcome the limiting effect of heme would be to increase intracellular heme levels. This requires a thorough insight into the biosynthetic pathway and its regulation. In this review, the heme biosynthetic pathway is discussed with regards to synthesis, regulation, and transport. Although the heme biosynthetic pathway is a highly conserved and tightly regulated pathway, the mode of regulation does not appear to be conserved among eukaryotes. However, common factors like feedback inhibition and regulation by heme, iron, and oxygen appear to be involved in regulation of the heme biosynthesis pathway in most organisms. Therefore, they are the initial targets to be investigated in Aspergillus niger.
DnaJ chaperones contribute to canalization.
Hughes, S.L. ; Vrinds, I. ; Roo, J. de ; Francke, C. ; Shimeld, S.M. ; Woollard, A. ; Sato, A.
2019, Article / Letter to editor (J Exp Zool A Ecol Integr Physiol, vol. 331, iss. 3, (2019), pp. 201-212)Canalization, an intrinsic robustness of development to external (environmental) or internal (genetic) perturbations, was first proposed over half a century ago. However, whether the robustness to environmental stress (environmental canalization [EC]) and to genetic variation (genetic canalization) are underpinned by the same molecular basis remains elusive. The recent discovery of the involvement of two endoplasmic reticulum (ER)-associated DnaJ genes in developmental buffering, orthologues of which are conserved across Metazoa, indicates that the role of ER-associated DnaJ genes might be conserved across the animal kingdom. To test this, we surveyed the ER-associated DnaJ chaperones in the nematode Caenorhabditis elegans. We then quantified the phenotype, in the form of variance and mean of seam cell counts, from RNA interference knockdown of DnaJs under three different temperatures. We find that seven out of eight ER-associated DnaJs are involved in either EC or microenvironmental canalization. Moreover, we also found two DnaJ genes not specifically associated with ER (DNAJC2/dnj-11 and DNAJA2/dnj-19) were involved in canalization. Protein expression pattern showed that these DnaJs are upregulated by heat stress, yet not all of them are expressed in the seam cells. Moreover, we found that most of the buffering DnaJs also control lifespan. We therefore concluded that a number of DnaJ chaperones, not limited to those associated with the ER, are involved in canalization as a part of the complex system that underlies development.
Development of an NGS data analysis strategy to identify the microbial population present in mushroom compost
Francke, C. ; Lamers, W.L. ; Bongers, J. ; Klijn, N. ; Horst, C. van der ; Lokman, B.C.
2016, Article in monograph or in proceedings (2016 19th ISMS Congress proceedings)
CiVi: circular genome visualization with unique features to analyze sequence elements.
Overmars, L. ; Hijum, S.A. van ; Siezen, R.J. ; Francke, C.
2015, Article / Letter to editor (Bioinformatics, vol. 31, iss. 17, (2015), pp. 2867-2869)We have developed CiVi, a user-friendly web-based tool to create custom circular maps to aid the analysis of microbial genomes and sequence elements. Sequence related data such as gene-name, COG class, PFAM domain, GC%, and subcellular location can be comprehensively viewed. Quantitative gene-related data (e.g. expression ratios or read counts) as well as predicted sequence elements (e.g. regulatory sequences) can be uploaded and visualized. CiVi accommodates the analysis of genomic elements by allowing a visual interpretation in the context of: (i) their genome-wide distribution, (ii) provided experimental data and (iii) the local orientation and location with respect to neighboring genes. CiVi thus enables both experts and non-experts to conveniently integrate public genome data with the results of genome analyses in circular genome maps suitable for publication. CONTACT: L.Overmars@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. AVAILABILITY AND IMPLEMENTATION: CiVi is freely available at http://civi.cmbi.ru.nl.
Analysis of the role of the A. niger aminolevulinic acid synthase (hemA) gene illustrates the difference between regulation of yeast and fungal heme and siroheme dependent pathways
Ram, Arthur F.J. ; Hondel, C.A. van den ; Lokman, B.C. ; Punt, P.J. ; Weert, S. de ; Franken, A.C.
2012, Article / Letter to editor (FEMS Microbiol Letters, vol. 335, iss. 2, (2012), pp. 104-112)To increase knowledge on haem biosynthesis in filamentous fungi like Aspergillus niger, pathway-specific gene expression in response to haem and haem intermediates was analysed. This analysis showed that iron, 5-aminolevulinic acid (ALA) and possibly haem control haem biosynthesis mostly via modulating expression of hemA [coding for 5-aminolevulinic acid synthase (ALAS)]. A hemA deletion mutant (?hemA) was constructed, which showed conditional lethality. Growth of ?hemA was supported on standard nitrate-containing media with ALA, but not by hemin. Growth of ?hemA could be sustained in the presence of hemin in combination with ammonium instead of nitrate as N-source. Our results suggest that a branch-off within the haem biosynthesis pathway required for sirohaem synthesis is responsible for lack of growth of ?hemA in media containing nitrate as sole N-source, because of the requirement of sirohaem for nitrate assimilation, as a cofactor of nitrite reductase. In contrast to the situation in Saccharomyces cerevisiae, cysteine, but not methionine, was found to further improve growth of ?hemA. These results demonstrate that A. niger can use exogenous hemin for its cellular processes. They also illustrate important differences in regulation of haem biosynthesis and in the role of haem and sirohaem in A. niger compared to S. cerevisiae.

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