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Dive into the research topics where Pascale Daran-Lapujade is active.

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Featured researches published by Pascale Daran-Lapujade.


Journal of Biological Chemistry | 2004

Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae A CHEMOSTAT CULTURE STUDY

Pascale Daran-Lapujade; Mickel L. A. Jansen; Jean-Marc Daran; Walter M. van Gulik; Johannes H. de Winde; Jack T. Pronk

In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growth-limiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms.


Proceedings of the National Academy of Sciences of the United States of America | 2007

The fluxes through glycolytic enzymes in Saccharomyces cerevisiae are predominantly regulated at posttranscriptional levels

Pascale Daran-Lapujade; Sergio Rossell; Walter M. van Gulik; Marijke A. H. Luttik; Marco J. L. de Groot; Monique Slijper; Albert J. R. Heck; Jean-Marc Daran; Johannes H. de Winde; Hans V. Westerhoff; Jack T. Pronk; Barbara M. Bakker

Metabolic fluxes may be regulated “hierarchically,” e.g., by changes of gene expression that adjust enzyme capacities (Vmax) and/or “metabolically” by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the ≈10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50–20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75–100% of the regulation of protein levels.


Molecular Systems Biology | 2006

When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation

M.T.A.P. Kresnowati; W.A. van Winden; Marinka J.H. Almering; A. ten Pierick; Cor Ras; Theo Knijnenburg; Pascale Daran-Lapujade; Jack T. Pronk; J. J. Heijnen; J.M. Daran

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro‐fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short‐term dynamics of the transcriptome revealed a remarkably fast decrease in the average half‐life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose‐induced regulation of gene expression.


Microbial Cell Factories | 2012

De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology.

Jurgen F. Nijkamp; Marcel van den Broek; Erwin Datema; Stefan de Kok; Lizanne Bosman; Marijke A. H. Luttik; Pascale Daran-Lapujade; Wanwipa Vongsangnak; Jens Nielsen; Wilbert H. M. Heijne; Paul Klaassen; Chris J. Paddon; Darren M. Platt; Peter Kötter; Roeland C. H. J. van Ham; Marcel J. T. Reinders; Jack T. Pronk; Dick de Ridder; Jean-Marc Daran

Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.


Fems Yeast Research | 2015

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.

Robert Mans; Harmen M. van Rossum; Melanie Wijsman; Antoon Backx; Niels G. A. Kuijpers; Marcel van den Broek; Pascale Daran-Lapujade; Jack T. Pronk; Antonius J. A. van Maris; Jean-Marc Daran

A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications. An open-source tool (http://yeastriction.tnw.tudelft.nl) is presented for identification of suitable Cas9 target sites in S. cerevisiae strains. A transformation strategy, using in vivo assembly of a guideRNA plasmid and subsequent genetic modification, was successfully implemented with high accuracies. An alternative strategy, using in vitro assembled plasmids containing two gRNAs, was used to simultaneously introduce up to six genetic modifications in a single transformation step with high efficiencies. Where previous studies mainly focused on the use of CRISPR/Cas9 for gene inactivation, we demonstrate the versatility of CRISPR/Cas9-based engineering of yeast by achieving simultaneous integration of a multigene construct combined with gene deletion and the simultaneous introduction of two single-nucleotide mutations at different loci. Sets of standardized plasmids, as well as the web-based Yeastriction target-sequence identifier and primer-design tool, are made available to the yeast research community to facilitate fast, standardized and efficient application of the CRISPR/Cas9 system.


Molecular Systems Biology | 2006

Proteome analysis of yeast response to various nutrient limitations

Annemieke Kolkman; Pascale Daran-Lapujade; Asier Fullaondo; Maurien Olsthoorn; Jack T. Pronk; Monique Slijper; Albert J. R. Heck

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and 15N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose‐repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β‐oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post‐transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology.


FEBS Journal | 2010

Measuring enzyme activities under standardized in vivo-like conditions for systems biology

K. van Eunen; J. Bouwman; Pascale Daran-Lapujade; J. Postmus; André B. Canelas; F.I.C. Mensonides; Rick Orij; I. Tuzun; J.M. van den Brink; Gertien J. Smits; W.M. van Gulik; Stanley Brul; J. J. Heijnen; J.H. de Winde; M. J. Teixeira de Mattos; Carsten Kettner; Jens Nielsen; Hans V. Westerhoff; Barbara M. Bakker

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme–kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme–kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113‐7D grown in aerobic glucose‐limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h−1. The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo‐like medium containing 300 mm potassium, 50 mm phosphate, 245 mm glutamate, 20 mm sodium, 2 mm free magnesium and 0.5 mm calcium, at a pH of 6.8. The Vmax values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme‐specific, optimal conditions.


Nature Communications | 2010

Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains

André B. Canelas; Nicola Harrison; Alessandro Fazio; Jie Zhang; Juha-Pekka Pitkänen; Joost van den Brink; Barbara M. Bakker; Lara Bogner; J. Bouwman; Juan I. Castrillo; Ayca Cankorur; Pramote Chumnanpuen; Pascale Daran-Lapujade; Duygu Dikicioglu; Karen van Eunen; Jennifer C. Ewald; Joseph J. Heijnen; Betul Kirdar; Ismo Mattila; F.I.C. Mensonides; Anja Niebel; Merja Penttilä; Jack T. Pronk; Matthias Reuss; Laura Salusjärvi; Uwe Sauer; David James Sherman; Martin Siemann-Herzberg; Hans V. Westerhoff; Johannes H. de Winde

The field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism.


Applied and Environmental Microbiology | 2009

Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

Léonie G.M. Boender; Erik de Hulster; Antonius J. A. van Maris; Pascale Daran-Lapujade; Jack T. Pronk

ABSTRACT Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h−1) was fitted with a 0.22-μm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h−1 (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g−1 of biomass h−1 calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h−1. This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, d-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae.


Fems Yeast Research | 2013

amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae

Daniel Solis-Escalante; Niels G. A. Kuijpers; Nadine Bongaerts; Irina Bolat; Lizanne Bosman; Jack T. Pronk; Jean-Marc Daran; Pascale Daran-Lapujade

Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, are therefore valuable assets in ambitious metabolic engineering programs. In the present work, the new recyclable dominant marker cassette amdSYM, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS), is presented. The amdSYM cassette confers S. cerevisiae the ability to use acetamide as sole nitrogen source. Direct repeats flanking the amdS gene allow for its efficient recombinative excision. As previously demonstrated in filamentous fungi, loss of the amdS marker cassette from S. cerevisiae can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in S. cerevisiae. Furthermore, using astute cassette design, amdSYM excision can be performed without leaving a scar or heterologous sequences in the targeted genome. The present work therefore demonstrates that amdSYM is a useful addition to the genetic engineering toolbox for Saccharomyces laboratory, wild, and industrial strains.

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Jack T. Pronk

Delft University of Technology

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Jean-Marc Daran

Delft University of Technology

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Johannes H. de Winde

Delft University of Technology

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Marcel van den Broek

Delft University of Technology

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Marijke A. H. Luttik

Delft University of Technology

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Erik de Hulster

Delft University of Technology

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Niels G. A. Kuijpers

Delft University of Technology

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Daniel Solis-Escalante

Delft University of Technology

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