1,047 results on '"Arnold, J M"'
Search Results
2. d-xylose accelerated death of pentose metabolizing Saccharomyces cerevisiae
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Nijland, Jeroen G., Zhang, Xiaohuan, and Driessen, Arnold J. M.
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- 2023
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3. Lateral membrane organization as target of an antimicrobial peptidomimetic compound
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Adéla Melcrová, Sourav Maity, Josef Melcr, Niels A. W. de Kok, Mariella Gabler, Jonne van der Eyden, Wenche Stensen, John S. M. Svendsen, Arnold J. M. Driessen, Siewert J. Marrink, and Wouter H. Roos
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Science - Abstract
Abstract Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.
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- 2023
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4. d-xylose accelerated death of pentose metabolizing Saccharomyces cerevisiae
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Jeroen G. Nijland, Xiaohuan Zhang, and Arnold J. M. Driessen
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d-xylose consumption ,ATP ,Xks1 expression ,Bioethanol ,Yeast ,Biotechnology ,TP248.13-248.65 ,Fuel ,TP315-360 - Abstract
Abstract Rapid and effective consumption of d-xylose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. Hence, heterologous d-xylose metabolic pathways have been introduced into S. cerevisiae. An effective solution is based on a xylose isomerase in combination with the overexpression of the xylulose kinase (Xks1) and all genes of the non-oxidative branch of the pentose phosphate pathway. Although this strain is capable of consuming d-xylose, growth inhibition occurs at higher d-xylose concentrations, even abolishing growth completely at 8% d-xylose. The decreased growth rates are accompanied by significantly decreased ATP levels. A key ATP-utilizing step in d-xylose metabolism is the phosphorylation of d-xylulose by Xks1. Replacement of the constitutive promoter of XKS1 by the galactose tunable promoter Pgal10 allowed the controlled expression of this gene over a broad range. By decreasing the expression levels of XKS1, growth at high d-xylose concentrations could be restored concomitantly with increased ATP levels and high rates of xylose metabolism. These data show that in fermentations with high d-xylose concentrations, too high levels of Xks1 cause a major drain on the cellular ATP levels thereby reducing the growth rate, ultimately causing substrate accelerated death. Hence, expression levels of XKS1 in S. cerevisiae needs to be tailored for the specific growth conditions and robust d-xylose metabolism.
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- 2023
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5. Combined roles of exporters in acetic acid tolerance in Saccharomyces cerevisiae
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Xiaohuan Zhang, Jeroen G. Nijland, and Arnold J. M. Driessen
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Acetic acid ,Acetate efflux ,Aqr1 ,Tpo2 ,Tpo3 ,Saccharomyces cerevisiae ,Biotechnology ,TP248.13-248.65 ,Fuel ,TP315-360 - Abstract
Abstract Acetic acid is a growth inhibitor generated during alcoholic fermentation and pretreatment of lignocellulosic biomass, a major feedstock to produce bioethanol. An understanding of the acetic acid tolerance mechanisms is pivotal for the industrial production of bioethanol. One of the mechanisms for acetic acid tolerance is transporter-mediated secretion where individual transporters have been implicated. Here, we deleted the transporters Aqr1, Tpo2, and Tpo3, in various combinations, to investigate their combined role in acetic acid tolerance. Single transporter deletions did not impact the tolerance at mild acetic acid stress (20 mM), but at severe stress (50 mM) growth was decreased or impaired. Tpo2 plays a crucial role in acetic acid tolerance, while the AQR1 deletion has a least effect on growth and acetate efflux. Deletion of both Tpo2 and Tpo3 enhanced the severe growth defects at 20 mM acetic acid concomitantly with a reduced rate of acetate secretion, while TPO2 and/or TPO3 overexpression in ∆tpo2∆tpo3∆ restored the tolerance. In the deletion strains, the acetate derived from sugar metabolism accumulated intracellularly, while gene transcription analysis suggests that under these conditions, ethanol metabolism is activated while acetic acid production is reduced. The data demonstrate that Tpo2 and Tpo3 together fulfill an important role in acetate efflux and the acetic acid response.
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- 2022
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6. The catalytic and structural basis of archaeal glycerophospholipid biosynthesis
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de Kok, Niels A. W. and Driessen, Arnold J. M.
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- 2022
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7. Combined roles of exporters in acetic acid tolerance in Saccharomyces cerevisiae
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Zhang, Xiaohuan, Nijland, Jeroen G., and Driessen, Arnold J. M.
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- 2022
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8. Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi
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László Mózsik, Riccardo Iacovelli, Roel A. L. Bovenberg, and Arnold J. M. Driessen
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secondary metabilites ,biosynthetic gene cluster ,synthetic biology ,synthetic transcriptional regulators ,fungal platform strains ,Biotechnology ,TP248.13-248.65 - Abstract
Filamentous fungi are highly productive cell factories, many of which are industrial producers of enzymes, organic acids, and secondary metabolites. The increasing number of sequenced fungal genomes revealed a vast and unexplored biosynthetic potential in the form of transcriptionally silent secondary metabolite biosynthetic gene clusters (BGCs). Various strategies have been carried out to explore and mine this untapped source of bioactive molecules, and with the advent of synthetic biology, novel applications, and tools have been developed for filamentous fungi. Here we summarize approaches aiming for the expression of endogenous or exogenous natural product BGCs, including synthetic transcription factors, assembly of artificial transcription units, gene cluster refactoring, fungal shuttle vectors, and platform strains.
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- 2022
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9. Membrane Adaptations and Cellular Responses of Sulfolobus acidocaldarius to the Allylamine Terbinafine
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Alka Rao, Niels A. W. de Kok, and Arnold J. M. Driessen
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GDGT ,archaea ,membrane ,Sulfolobus ,isoprenoids ,caldariellaquinone ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive. Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation.
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- 2023
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10. Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae
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Ivan Rodrigo Wolf, Lucas Farinazzo Marques, Lauana Fogaça de Almeida, Lucas Cardoso Lázari, Leonardo Nazário de Moraes, Luiz Henrique Cardoso, Camila Cristina de Oliveira Alves, Rafael Takahiro Nakajima, Amanda Piveta Schnepper, Marjorie de Assis Golim, Thais Regiani Cataldi, Jeroen G. Nijland, Camila Moreira Pinto, Matheus Naia Fioretto, Rodrigo Oliveira Almeida, Arnold J. M. Driessen, Rafael Plana Simōes, Mônica Veneziano Labate, Rejane Maria Tommasini Grotto, Carlos Alberto Labate, Ary Fernandes Junior, Luis Antonio Justulin, Rafael Luiz Buogo Coan, Érica Ramos, Fabiana Barcelos Furtado, Cesar Martins, and Guilherme Targino Valente
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omics ,data integration ,systems biology ,lncRNAs ,lncRNA–protein interactions ,membraneless organelles ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.
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- 2023
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11. Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis.
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Bailoni, Eleonora, Patiño-Ruiz, Miyer F., Stan, Andreea R., Schuurman-Wolters, Gea K., Exterkate, Marten, Driessen, Arnold J. M., and Poolman, Bert
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- 2024
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12. CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
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László Mózsik, Mirthe Hoekzema, Niels A. W. de Kok, Roel A. L. Bovenberg, Yvonne Nygård, and Arnold J. M. Driessen
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Medicine ,Science - Abstract
Abstract Filamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.
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- 2021
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13. Cellular dynamics of the SecA ATPase at the single molecule level
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Anne-Bart Seinen, Dian Spakman, Antoine M. van Oijen, and Arnold J. M. Driessen
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Medicine ,Science - Abstract
Abstract In bacteria, the SecA ATPase provides the driving force for protein secretion via the SecYEG translocon. While the dynamic interplay between SecA and SecYEG in translocation is widely appreciated, it is not clear how SecA associates with the translocon in the crowded cellular environment. We use super-resolution microscopy to directly visualize the dynamics of SecA in Escherichia coli at the single-molecule level. We find that SecA is predominantly associated with and evenly distributed along the cytoplasmic membrane as a homodimer, with only a minor cytosolic fraction. SecA moves along the cell membrane as three distinct but interconvertible diffusional populations: (1) A state loosely associated with the membrane, (2) an integral membrane form, and (3) a temporarily immobile form. Disruption of the proton-motive-force, which is essential for protein secretion, re-localizes a significant portion of SecA to the cytoplasm and results in the transient location of SecA at specific locations at the membrane. The data support a model in which SecA diffuses along the membrane surface to gain access to the SecYEG translocon.
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- 2021
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14. Cellular dynamics of the SecA ATPase at the single molecule level
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Seinen, Anne-Bart, Spakman, Dian, van Oijen, Antoine M., and Driessen, Arnold J. M.
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- 2021
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15. Author Correction: CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
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Mózsik, László, Hoekzema, Mirthe, de Kok, Niels A. W., Bovenberg, Roel A. L., Nygård, Yvonne, and Driessen, Arnold J. M.
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- 2021
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16. CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
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Mózsik, László, Hoekzema, Mirthe, de Kok, Niels A. W., Bovenberg, Roel A. L., Nygård, Yvonne, and Driessen, Arnold J. M.
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- 2021
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17. Synthetic control devices for gene regulation in Penicillium chrysogenum
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László Mózsik, Zsófia Büttel, Roel A. L. Bovenberg, Arnold J. M. Driessen, and Yvonne Nygård
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Synthetic expression system ,Gene regulation ,Penicillium chrysogenum ,Synthetic gene cluster ,Secondary metabolite production ,Hybrid transcription factor ,Microbiology ,QR1-502 - Abstract
Abstract Background Orthogonal, synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster of Neurospora crassa is fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system. Results The control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, upstream activating sequences, and the affinity of the DNA binding domain of the transcription factor to the upstream activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin. Conclusions The characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.
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- 2019
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18. Synthetic Minimal Cell: Self-Reproduction of the Boundary Layer
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Marten Exterkate and Arnold J. M. Driessen
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Chemistry ,QD1-999 - Published
- 2019
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19. Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane
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Caforio, Antonella, Siliakus, Melvin F., Exterkate, Marten, Jain, Samta, Jumde, Varsha R., Andringa, Ruben L. H., Kengen, Servé W. M., Minnaard, Adriaan J., Driessen, Arnold J. M., and van der Oost, John
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- 2018
20. Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin
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Sietske Grijseels, Carsten Pohl, Jens Christian Nielsen, Zahida Wasil, Yvonne Nygård, Jens Nielsen, Jens C. Frisvad, Kristian Fog Nielsen, Mhairi Workman, Thomas Ostenfeld Larsen, Arnold J. M. Driessen, and Rasmus John Normand Frandsen
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Penicillium decumbens ,Calbistrin ,Secondary metabolite ,Decalin ,Polyketide ,Biosynthesis ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. Results A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. Conclusion Here we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.
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- 2018
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21. A Penicillium rubens platform strain for secondary metabolite production
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Pohl, Carsten, Polli, Fabiola, Schütze, Tabea, Viggiano, Annarita, Mózsik, László, Jung, Sascha, de Vries, Maaike, Bovenberg, Roel A. L., Meyer, Vera, and Driessen, Arnold J. M.
- Published
- 2020
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22. Biochemical characterization of the Nocardia lactamdurans ACV synthetase.
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Riccardo Iacovelli, Reto D Zwahlen, Roel A L Bovenberg, and Arnold J M Driessen
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Medicine ,Science - Abstract
The L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS) is a nonribosomal peptide synthetase (NRPS) that fulfills a crucial role in the synthesis of β-lactams. Although some of the enzymological aspects of this enzyme have been elucidated, its large size, at over 400 kDa, has hampered heterologous expression and stable purification attempts. Here we have successfully overexpressed the Nocardia lactamdurans ACVS in E. coli HM0079. The protein was purified to homogeneity and characterized for tripeptide formation with a focus on the substrate specificity of the three modules. The first L-α-aminoadipic acid-activating module is highly specific, whereas the modules for L-cysteine and L-valine are more promiscuous. Engineering of the first module of ACVS confirmed the strict specificity observed towards its substrate, which can be understood in terms of the non-canonical peptide bond position.
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- 2020
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23. Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications
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Jeroen G. Nijland and Arnold J. M. Driessen
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pentose transport ,D-xylose ,L-arabinose ,yeast ,bioethanol ,Biotechnology ,TP248.13-248.65 - Abstract
Lignocellulosic biomass yields after hydrolysis, besides the hexose D-glucose, D-xylose, and L-arabinose as main pentose sugars. In second generation bioethanol production utilizing the yeast Saccharomyces cerevisiae, it is critical that all three sugars are co-consumed to obtain an economically feasible and robust process. Since S. cerevisiae is unable to metabolize pentose sugars, metabolic pathway engineering has been employed to introduce the respective pathways for D-xylose and L-arabinose metabolism. However, S. cerevisiae lacks specific pentose transporters, and these sugars enter the cell with low affinity via glucose transporters of the Hxt family. Therefore, in the presence of D-glucose, utilization of D-xylose and L-arabinose is poor as the Hxt transporters prefer D-glucose. To solve this problem, heterologous expression of pentose transporters has been attempted but often with limited success due to poor expression and stability, and/or low turnover. A more successful approach is the engineering of the endogenous Hxt transporter family and evolutionary selection for D-glucose insensitive growth on pentose sugars. This has led to the identification of a critical and conserved asparagine residue in Hxt transporters that, when mutated, reduces the D-glucose affinity while leaving the D-xylose affinity mostly unaltered. Likewise, mutant Gal2 transporter have been selected supporting specific uptake of L-arabinose. In fermentation experiments, the transporter mutants support efficient uptake and consumption of pentose sugars, and even co-consumption of D-xylose and D-glucose when used at industrial concentrations. Further improvements are obtained by interfering with the post-translational inactivation of Hxt transporters at high or low D-glucose concentrations. Transporter engineering solved major limitations in pentose transport in yeast, now allowing for co-consumption of sugars that is limited only by the rates of primary metabolism. This paves the way for a more economical second-generation biofuels production process.
- Published
- 2020
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24. The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae
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Jasmine M. Bracher, Maarten D. Verhoeven, H. Wouter Wisselink, Barbara Crimi, Jeroen G. Nijland, Arnold J. M. Driessen, Paul Klaassen, Antonius J. A. van Maris, Jean-Marc G. Daran, and Jack T. Pronk
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Penicillium ,Transcriptome ,Sugar transport ,Proton symport ,l-Arabinose transporter ,Second-generation bioethanol ,Fuel ,TP315-360 ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (K m = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10−3 and 1.8 g L−1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L−1 l-arabinose and 20 g L−1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.
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- 2018
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25. Announcing Molecular Biomedicine
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Driessen, Arnold J. M. and Wei, Yu-quan
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- 2020
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26. Synthetic control devices for gene regulation in Penicillium chrysogenum
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Mózsik, László, Büttel, Zsófia, Bovenberg, Roel A. L., Driessen, Arnold J. M., and Nygård, Yvonne
- Published
- 2019
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27. Author Correction: CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
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László Mózsik, Mirthe Hoekzema, Niels A. W. de Kok, Roel A. L. Bovenberg, Yvonne Nygård, and Arnold J. M. Driessen
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Medicine ,Science - Published
- 2021
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28. Heterologous Naringenin Production in the Filamentous Fungus Penicillium rubens.
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Peng, Bo, Dai, Lin, Iacovelli, Riccardo, Driessen, Arnold J. M., and Haslinger, Kristina
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- 2023
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29. Announcing Molecular Biomedicine
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Arnold J. M. Driessen and Yu-quan Wei
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Medicine - Published
- 2020
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30. Engineering of the Filamentous Fungus Penicillium chrysogenum as Cell Factory for Natural Products
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Fernando Guzmán-Chávez, Reto D. Zwahlen, Roel A. L. Bovenberg, and Arnold J. M. Driessen
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Penicillium chrysogenum ,natural products ,nonribosomal peptides ,polyketides ,gene activation ,biosynthetic gene clusters ,Microbiology ,QR1-502 - Abstract
Penicillium chrysogenum (renamed P. rubens) is the most studied member of a family of more than 350 Penicillium species that constitute the genus. Since the discovery of penicillin by Alexander Fleming, this filamentous fungus is used as a commercial β-lactam antibiotic producer. For several decades, P. chrysogenum was subjected to a classical strain improvement (CSI) program to increase penicillin titers. This resulted in a massive increase in the penicillin production capacity, paralleled by the silencing of several other biosynthetic gene clusters (BGCs), causing a reduction in the production of a broad range of BGC encoded natural products (NPs). Several approaches have been used to restore the ability of the penicillin production strains to synthetize the NPs lost during the CSI. Here, we summarize various re-activation mechanisms of BGCs, and how interference with regulation can be used as a strategy to activate or silence BGCs in filamentous fungi. To further emphasize the versatility of P. chrysogenum as a fungal production platform for NPs with potential commercial value, protein engineering of biosynthetic enzymes is discussed as a tool to develop de novo BGC pathways for new NPs.
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- 2018
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31. Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin
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Grijseels, Sietske, Pohl, Carsten, Nielsen, Jens Christian, Wasil, Zahida, Nygård, Yvonne, Nielsen, Jens, Frisvad, Jens C., Nielsen, Kristian Fog, Workman, Mhairi, Larsen, Thomas Ostenfeld, Driessen, Arnold J. M., and Frandsen, Rasmus John Normand
- Published
- 2018
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32. Prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw foods in Peshawar, Pakistan
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Taj Ali Khan, Jeroen G. Nijland, Hazir Rahman, Iqbal Nisa, Rafiullah, Anwar Ali, Muhammad Qasim, Abdullah Jalal, Munazza Raza Mirza, Arnold J. M. Driessen, Mirza Ali Khan, and Molecular Microbiology
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Shigellosis ,Veterinary medicine ,030309 nutrition & dietetics ,Water contamination ,Water source ,Shigella flexneri ,Shigella species ,03 medical and health sciences ,0404 agricultural biotechnology ,Raw Foods ,Risk Factors ,RNA, Ribosomal, 16S ,Prevalence ,medicine ,Humans ,Pakistan ,Phylogeny ,Dysentery, Bacillary ,0303 health sciences ,biology ,Potential risk ,Drinking Water ,04 agricultural and veterinary sciences ,Raw milk ,medicine.disease ,biology.organism_classification ,040401 food science ,16s rrna gene sequencing ,Food Science - Abstract
This study was designed to investigate the prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw food samples in Peshawar, Pakistan. A total of 1,020 different samples were collected from various areas of Peshawar between January 2016 and May 2017, followed by identification of S. flexneri through biochemical, serological, and 16S rRNA gene sequencing. Potential risk factors associated with the development and spreading of S. flexneri infection were also investigated. Overall, 45 (4.41%) samples were positive for Shigella species. Among these samples, the predominant species was S. flexneri (n = 44) followed by S. boydii (n = 1). Interestingly, S. sonnei and S. dysenteriae isolates were not found in any sample. The isolation rate of S. flexneri in drinking water samples, market raw milk, and fruits/vegetables from Peshawar were 6.47%, 3.5%, and 2.9%, respectively. The phylogenetic reconstruction showed genetic diversity among three clades, as clades I and II have isolates of S. flexneri that were circulating within the drinking water, milk, fruits/vegetables, while clade III isolates were recovered from milk samples. Most of S. flexneri were detected in June to September. Potential risk factors of S. flexneri were water sources contaminated by toilet wastes (p = 0.04), surface water drainage (p = 0.0002), hospital wastes (p = 0.01), unhygienic handling (p < 0.05), and transportation of raw food (p = 0.04). In conclusion, S. flexneri isolates of closely related lineage originating from non-clinical samples might be associated with an increased human risk to shigellosis in Pakistan, as significant numbers of S. flexneri were observed in the drinking water and retail raw food samples. PRACTICAL APPLICATION: This study demonstrated the presence of S. flexneri in drinking water and retail raw food samples which seem to possess a serious threat to public health. Potential sources of food and water contamination should properly be monitored by public health authorities to reduce cases of shigellosis.
- Published
- 2021
33. Lateral membrane organization as target of an antimicrobial peptidomimetic compound.
- Author
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Melcrová, Adéla, Maity, Sourav, Melcr, Josef, de Kok, Niels A. W., Gabler, Mariella, van der Eyden, Jonne, Stensen, Wenche, Svendsen, John S. M., Driessen, Arnold J. M., Marrink, Siewert J., and Roos, Wouter H.
- Subjects
BACTERIAL cell walls ,BENZALKONIUM chloride ,MOLECULAR dynamics ,DRUG resistance in microorganisms ,STAPHYLOCOCCUS aureus - Abstract
Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes. The mechanism of action of the antibacterial tripeptide AMC-109 is unclear. Here, Melcrová et al. show that AMC-109 self-assembles into stable aggregates with a cationic surface, and then individual peptides insert into the bacterial membrane and disrupt membrane nanodomains, thus affecting membrane function without forming pores. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
34. The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae
- Author
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Bracher, Jasmine M., Verhoeven, Maarten D., Wisselink, H. Wouter, Crimi, Barbara, Nijland, Jeroen G., Driessen, Arnold J. M., Klaassen, Paul, van Maris, Antonius J. A., Daran, Jean-Marc G., and Pronk, Jack T.
- Published
- 2018
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35. In Vivo Study of the Sorbicillinoid Gene Cluster in Trichoderma reesei
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Christian Derntl, Fernando Guzmán-Chávez, Thiago M. Mello-de-Sousa, Hans-Jürgen Busse, Arnold J. M. Driessen, Robert L. Mach, and Astrid R. Mach-Aigner
- Subjects
sorbicillinoids ,sorbicillinol ,5-hydroxyvertinolide ,Trichoderma reesei ,Acremonium chrysogenum ,Penicillium chrysogenum ,Microbiology ,QR1-502 - Abstract
Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2′,3′-dihydrosorbicillin to sorbicillinol and 2′,3′-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2′,3′-dihydrosorbicillin and 2′,3′-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.
- Published
- 2017
- Full Text
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36. The role of the N-terminal amphipathic helix in bacterial YidC
- Author
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Nass, Karol J, Ilie, Ioana M, Saller, Manfred J, Driessen, Arnold J M, Caflisch, Amedeo, Kammerer, Richard A, Li, Xiaodan, Molecular Microbiology, University of Zurich, and Ilie, Ioana M
- Subjects
1303 Biochemistry ,Protein Conformation ,Cell Membrane ,Biophysics ,Membrane Transport Proteins ,610 Medicine & health ,Cell Biology ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Biochemistry ,1307 Cell Biology ,Bacterial Proteins ,10019 Department of Biochemistry ,570 Life sciences ,biology ,Thermotoga maritima ,1304 Biophysics - Abstract
The evolutionary conserved YidC is a unique dual-function membrane protein that adopts insertase and chaperone conformations. The N-terminal helix of Escherichia coli YidC functions as an uncleaved signal sequence and is important for membrane insertion and interaction with the Sec translocon. Here, we report the first crystal structure of Thermotoga maritima YidC (TmYidC) including the N-terminal amphipathic helix (N-AH) (PDB ID: 6Y86). Molecular dynamics simulations show that N-AH lies on the periplasmic side of the membrane bilayer forming an angle of about 15° with the membrane surface. Our functional studies suggest a role of N-AH for the species-specific interaction with the Sec translocon. The reconstitution data and the superimposition of TmYidC with known YidC structures suggest an active insertase conformation for YidC. Molecular dynamics (MD) simulations of TmYidC provide evidence that N-AH acts as a membrane recognition helix for the YidC insertase and highlight the flexibility of the C1 region underlining its ability to switch between insertase and chaperone conformations. A structure-based model is proposed to rationalize how YidC performs the insertase and chaperone functions by re-positioning of N-AH and the other structural elements.
- Published
- 2022
37. Membrane Adaptations and Cellular Responses of Sulfolobus acidocaldarius to the Allylamine Terbinafine.
- Author
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Rao, Alka, de Kok, Niels A. W., and Driessen, Arnold J. M.
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TERBINAFINE ,ETHER lipids ,CELL membranes ,DEVIATORIC stress (Engineering) ,MEMBRANE lipids ,ISOPENTENOIDS ,CELL compartmentation ,BILAYER lipid membranes - Abstract
Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive. Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
38. Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae.
- Author
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Wolf, Ivan Rodrigo, Marques, Lucas Farinazzo, de Almeida, Lauana Fogaça, Lázari, Lucas Cardoso, de Moraes, Leonardo Nazário, Cardoso, Luiz Henrique, Alves, Camila Cristina de Oliveira, Nakajima, Rafael Takahiro, Schnepper, Amanda Piveta, Golim, Marjorie de Assis, Cataldi, Thais Regiani, Nijland, Jeroen G., Pinto, Camila Moreira, Fioretto, Matheus Naia, Almeida, Rodrigo Oliveira, Driessen, Arnold J. M., Simōes, Rafael Plana, Labate, Mônica Veneziano, Grotto, Rejane Maria Tommasini, and Labate, Carlos Alberto
- Subjects
SACCHAROMYCES cerevisiae ,LINCRNA ,ETHANOL ,RIBOSOMAL RNA ,DATA integration ,PROTEIN metabolism - Abstract
Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
39. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter
- Author
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Yang, Nuan and Driessen, Arnold J. M.
- Published
- 2015
- Full Text
- View/download PDF
40. The bacterial Sec-translocase: structure and mechanism
- Author
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Lycklama a Nijeholt, Jelger A. and Driessen, Arnold J. M.
- Published
- 2012
41. Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules
- Author
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Bonardi, Francesco, Halza, Erik, Walko, Martin, Du Plessis, François, Nouwen, Nico, Feringa, Ben L., Driessen, Arnold J. M., and Randall, Linda L.
- Published
- 2011
42. Synthetic control devices for gene regulation in Penicillium chrysogenum
- Author
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Zsófia Büttel, Yvonne Nygård, László Mózsik, Roel A. L. Bovenberg, Arnold J. M. Driessen, and Molecular Microbiology
- Subjects
Models, Molecular ,STRAIN ,Genes, Fungal ,lcsh:QR1-502 ,PROTEIN ,Bioengineering ,ORGANIZATION ,Penicillium chrysogenum ,Applied Microbiology and Biotechnology ,lcsh:Microbiology ,Neurospora crassa ,Fungal Proteins ,03 medical and health sciences ,Upstream activating sequence ,CORE PROMOTERS ,TET-ON ,Gene Expression Regulation, Fungal ,Gene cluster ,Transgenes ,Secondary metabolite production ,Hybrid transcription factor ,Gene ,Transcription factor ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,biology ,Organisms, Genetically Modified ,PARTS ,030306 microbiology ,Chemistry ,Research ,EXPRESSION SYSTEM ,CLUSTER ,DNA-binding domain ,biology.organism_classification ,Q-SYSTEM ,Biosynthetic Pathways ,Gene regulation ,Synthetic expression system ,Biochemistry ,Synthetic gene cluster ,Biotechnology ,Transcription Factors - Abstract
BackgroundOrthogonal, synthetic control devices were developed forPenicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster ofNeurospora crassais fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system.ResultsThe control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, upstream activating sequences, and the affinity of the DNA binding domain of the transcription factor to the upstream activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin.ConclusionsThe characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.
- Published
- 2019
43. Modular synthetic biology toolkit for filamentous fungi
- Author
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Roel A. L. Bovenberg, Carsten Pohl, Yvonne Nygård, Arnold J. M. Driessen, Vera Meyer, László Mózsik, Molecular Microbiology, and Groningen Biomolecular Sciences and Biotechnology
- Subjects
Letter ,inducible promoter ,Genetic Vectors ,Biomedical Engineering ,Computational biology ,Biology ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,03 medical and health sciences ,Synthetic biology ,synthetic biology toolkit ,Shuttle vector ,ddc:570 ,Transcriptional regulation ,Modular Cloning ,CRISPR ,transcriptional regulation ,Cloning, Molecular ,Promoter Regions, Genetic ,hybrid transcription factor ,030304 developmental biology ,Cloning ,0303 health sciences ,030306 microbiology ,business.industry ,filamentous fungi ,Fungi ,Promoter ,General Medicine ,Modular design ,Fusion protein ,Synthetic Biology ,CRISPR-Cas Systems ,Genetic Engineering ,business ,Plasmids - Abstract
Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.
- Published
- 2021
44. Direct Observation of Chaperone-Induced Changes in a Protein Folding Pathway
- Author
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Bechtluft, Philipp, van Leeuwen, Ruud G. H., Tyreman, Matthew, Tomkiewicz, Danuta, Nouwen, Nico, Tepper, Harald L., Driessen, Arnold J. M., and Tans, Sander J.
- Published
- 2007
- Full Text
- View/download PDF
45. Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome
- Author
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Sonja-Verena Albers and Arnold J. M. Driessen
- Subjects
Microbiology ,QR1-502 - Abstract
The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.
- Published
- 2008
- Full Text
- View/download PDF
46. A versatile method to separate complex lipid mixtures using 1-butanol as eluent in a reverse-phase UHPLC-ESI-MS system
- Author
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Arnold J. M. Driessen, Marten Exterkate, Niels A W de Kok, Adriaan J. Minnaard, Ruben L. H. Andringa, Molecular Microbiology, and Chemical Biology 2
- Subjects
Sulfolobus acidocaldarius ,Spectrometry, Mass, Electrospray Ionization ,Chromatography ,Electrospray ionization ,Organic Chemistry ,Phospholipid ,Cell Biology ,Lipidome ,Biochemistry ,Lipids ,chemistry.chemical_compound ,Column chromatography ,1-Butanol ,chemistry ,Glycerophospholipid ,Cardiolipins ,Lipidomics ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Chromatography, High Pressure Liquid - Abstract
Simple, robust and versatile LC-MS based methods add to the rapid assessment of the lipidome of biological cells. Here we present a versatile RP-UHPLC-MS method using 1-butanol as the eluent, specifically designed to separate different highly hydrophobic lipids. This method is capable of separating different lipid classes of glycerophospholipid standards, in addition to phospholipids of the same class with a different acyl chain composition. The versatility of this method was demonstrated through analysis of lipid extracts of the bacterium Escherichia coli and the archaeon Sulfolobus acidocaldarius. In contrast to 2-propanol-based methods, the 1-butanol-based mobile phase is capable of eluting highly hydrophobic analytes such as cardiolipins, tetraether lipids and mycolic acids during the gradient instead of the isocratic purge phase, resulting in an enhanced separation of cardiolipins and extending the analytical range for RPLC.
- Published
- 2021
47. Deletion of cdvB paralogous genes of Sulfolobus acidocaldarius impairs cell division
- Author
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Yang, Nuan and Driessen, Arnold J. M.
- Published
- 2014
- Full Text
- View/download PDF
48. In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis.
- Author
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Irfan Prabudiansyah, Ilja Kusters, and Arnold J M Driessen
- Subjects
Medicine ,Science - Abstract
The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.
- Published
- 2015
- Full Text
- View/download PDF
49. Binding of the Lactococcal Drug Dependent Transcriptional Regulator LmrR to Its Ligands and Responsive Promoter Regions.
- Author
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Jan Pieter van der Berg, Pramod Kumar Madoori, Amalina Ghaisani Komarudin, Andy-Mark Thunnissen, and Arnold J M Driessen
- Subjects
Medicine ,Science - Abstract
The heterodimeric ABC transporter LmrCD from Lactococcus lactis is able to extrude several different toxic compounds from the cell, fulfilling a role in the intrinsic and induced drug resistance. The expression of the lmrCD genes is regulated by the multi-drug binding repressor LmrR, which also binds to its own promoter to autoregulate its own expression. Previously, we reported the crystal structure of LmrR in the presence and absence of the drugs Hoechst 33342 and daunomycin. Analysis of the mechanism how drugs control the repressor activity of LmrR is impeded by the fact that these drugs also bind to DNA. Here we identified, using X-ray crystallography and fluorescence, that riboflavin binds into the drug binding cavity of LmrR, adopting a similar binding mode as Hoechst 33342 and daunomycin. Microscale thermophoresis was employed to quantify the binding affinity of LmrR to its responsive promoter regions and to evaluate the cognate site of LmrR in the lmrCD promoter region. Riboflavin reduces the binding affinity of LmrR for the promoter regions. Our results support a model wherein drug binding to LmrR relieves the LmrR dependent repression of the lmrCD genes.
- Published
- 2015
- Full Text
- View/download PDF
50. Biosynthesis of archaeal membrane ether lipids
- Author
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Samta eJain, Antonella eCaforio, and Arnold J. M. Driessen
- Subjects
Archaea ,biosynthesis ,isoprenoids ,ether lipids ,lipid divide ,Microbiology ,QR1-502 - Abstract
A vital function of the cell membrane in all living organism is to maintain the membrane permeability barrier and fluidity. The composition of the phospholipid bilayer is distinct in archaea when compared to bacteria and eukarya. In archaea, isoprenoid hydrocarbon side chains are linked via an ether bond to the sn-glycerol-1-phosphate backbone. In bacteria and eukarya on the other hand, fatty acid side chains are linked via an ester bond to the sn-glycerol-3-phosphate backbone. The polar head groups are globally shared in the three domains of life. The unique membrane lipids of archaea have been implicated not only in the survival and adaptation of the organisms to extreme environments but also to form the basis of the membrane composition of the last universal common ancestor (LUCA). In nature, a diverse range of archaeal lipids is found, the most common are the diether (or archaeol) and the tetraether (or caldarchaeol) lipids that form a monolayer. Variations in chain length, cyclization and other modifications lead to diversification of these lipids. The biosynthesis of these lipids is not yet well understood however progress in the last decade has led to a comprehensive understanding of the biosynthesis of archaeol. This review describes the current knowledge of the biosynthetic pathway of archaeal ether lipids; insights on the stability and robustness of archaeal lipid membranes; and evolutionary aspects of the lipid divide and the last universal common ancestor LUCA. It examines recent advances made in the field of pathway reconstruction in bacteria.
- Published
- 2014
- Full Text
- View/download PDF
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