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Draft genome sequence of the Saccharomyces cerevisiae SpyCas9 expressing strain IMX2600, a laboratory and platform strain from the CEN.PK lineage for cell-factory research

van den Broek, M.A. (author), Ortiz Merino, R.A. (author), Bennis, N.X. (author), Wronska, A.K. (author), Else-Hassing, J. (author), Daran-Lapujade, P.A.S. (author), Daran, J.G. (author)

The biobased-economy aims to create a circular biotechnology ecosystem to transition from a fossil fuel-based to a sustainable industry based on biomass. For this, new microbial cell-factories are essential. We present the draft genome of the CEN.PK-derived Saccharomyces cerevisiae SpyCas9 expressing strain (IMX2600), that serve as chassis of...

journal article 2024

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Proteome Dynamics During Transition From Exponential to Stationary Phase Under Aerobic and Anaerobic Conditions in Yeast

den Ridder, M.J. (author), van den Brandeler, Wiebeke (author), Altiner, Meryem (author), Daran-Lapujade, P.A.S. (author), Pabst, Martin (author)

The yeast Saccharomyces cerevisiae is a widely-used eukaryotic model organism and a promising cell factory for industry. However, despite decades of research, the regulation of its metabolism is not yet fully understood, and its complexity represents a major challenge for engineering and optimizing biosynthetic routes. Recent studies have...

journal article 2023

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Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae reveals condition-dependent intron abundance

Koster, C.C. (author), Kleefeldt, Askar A. (author), van den Broek, M.A. (author), Luttik, M.A.H. (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene...

journal article 2023

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Top-Down, Knowledge-Based Genetic Reduction of Yeast Central Carbon Metabolism

Postma, E.D. (author), Couwenberg, Lucas G.F. (author), van Roosmalen, Roderick N. (author), Geelhoed, J. (author), de Groot, P.A. (author), Daran-Lapujade, P.A.S. (author)

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterized by a mosaic genome configuration with substantial apparent genetic redundancy. This apparent redundancy raises questions about the evolutionary driving force for genomic fixation of “minor” paralogs and complicates modular and...

journal article 2022

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Full humanization of the glycolytic pathway in Saccharomyces cerevisiae

Boonekamp, F.J. (author), Knibbe, E. (author), Vieira Lara, M.A. (author), Wijsman, M. (author), Luttik, M.A.H. (author), van Eunen, Karen (author), den Ridder, M.J. (author), Pabst, Martin (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single...

journal article 2022

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Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

Postma, E.D. (author), Else-Hassing, J. (author), Mangkusaputra, Venda (author), Geelhoed, J. (author), de la Torre, P. (author), van den Broek, M.A. (author), Mooiman, C. (author), Pabst, Martin (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo...

journal article 2022

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Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies

Elsemman, Ibrahim E. (author), Rodriguez Prado, E.A. (author), Grigaitis, Pranas (author), Garcia Albornoz, Manuel (author), Harman, Victoria (author), Holman, Stephen W. (author), van Heerden, Johan (author), Bisschops, M.M.M. (author), Daran-Lapujade, P.A.S. (author)

When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of...

journal article 2022

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A systematic evaluation of yeast sample preparation protocols for spectral identifications, proteome coverage and post-isolation modifications

den Ridder, M.J. (author), Knibbe, E. (author), van den Brandeler, Wiebeke (author), Daran-Lapujade, P.A.S. (author), Pabst, Martin (author)

The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols. In particular when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory Saccharomyces cerevisiae. Here, we...

journal article 2022

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Synthetic Genomics From a Yeast Perspective

Koster, C.C. (author), Postma, E.D. (author), Knibbe, E. (author), Cleij, C.V. (author), Daran-Lapujade, P.A.S. (author)

Synthetic Genomics focuses on the construction of rationally designed chromosomes and genomes and offers novel approaches to study biology and to construct synthetic cell factories. Currently, progress in Synthetic Genomics is hindered by the inability to synthesize DNA molecules longer than a few hundred base pairs, while the size of the...

review 2022

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A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

Postma, E.D. (author), Dashko, S. (author), van Breemen, Lars (author), Taylor Parkins, S.K. (author), van den Broek, M.A. (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities....

journal article 2021

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GEL DNA: A Cloning-and Polymerase Chain Reaction-Free Method for CRISPR-Based Multiplexed Genome Editing

Randazzo, P. (author), Bennis, N.X. (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

Even for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR-Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events, and diverse approaches for multiplex genome editing have been described in the...

journal article 2021

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Design and Experimental Evaluation of a Minimal, Innocuous Watermarking Strategy to Distinguish Near-Identical DNA and RNA Sequences

Boonekamp, F.J. (author), Dashko, S. (author), Duiker, Donna (author), Gehrmann, T. (author), van den Broek, M.A. (author), den Ridder, M.J. (author), Pabst, Martin (author), Robert, Vincent (author), Abeel, T.E.P.M.F. (author), Postma, E.D. (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavors, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well...

journal article 2020

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A proteome-integrated, carbon source dependent genetic regulatory network in: Saccharomyces cerevisiae

Garcia-Albornoz, M. (author), Holman, S. W. (author), Antonisse, T. (author), Daran-Lapujade, P.A.S. (author), Teusink, B. (author), Beynon, R. J. (author), Hubbard, S. J. (author)

Integrated regulatory networks can be powerful tools to examine and test properties of cellular systems, such as modelling environmental effects on the molecular bioeconomy, where protein levels are altered in response to changes in growth conditions. Although extensive regulatory pathways and protein interaction data sets exist which...

journal article 2020

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Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO₂ levels

Hakkaart, X.D.V. (author), Liu, Y. (author), Hulst, Mandy (author), el Masoudi, Anissa (author), Peuscher, Eveline (author), Pronk, J.T. (author), van Gulik, W.M. (author), Daran-Lapujade, P.A.S. (author)

Engineered strains of Saccharomyces cerevisiae are used for industrial production of succinic acid. Optimal process conditions for dicarboxylic-acid yield and recovery include slow growth, low pH, and high CO₂. To quantify and understand how these process parameters affect yeast physiology, this study investigates individual and...

journal article 2019

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Multiplex genome editing of microorganisms using CRISPR-Cas

Adiego-Pérez, Belén (author), Randazzo, P. (author), Daran, J.G. (author), Verwaal, René (author), Roubos, Johannes A. (author), Daran-Lapujade, P.A.S. (author), Van Der Oost, John (author)

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made...

review 2019

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The Genetic Makeup and Expression of the Glycolytic and Fermentative Pathways Are Highly Conserved Within the Saccharomyces Genus

Boonekamp, F.J. (author), Dashko, S. (author), van den Broek, M.A. (author), Gehrmann, T. (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

The ability of the yeast Saccharomyces cerevisiae to convert glucose, even in the presence of oxygen, via glycolysis and the fermentative pathway to ethanol has played an important role in its domestication. Despite the extensive knowledge on these pathways in S. cerevisiae, relatively little is known about their genetic makeup in other...

journal article 2018

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A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains

Wijsman, M. (author), Swiat, M.A. (author), Marques, W.L. (author), Hettinga, Johanna K. (author), van den Broek, M.A. (author), Torre Cortés, Pilar de la (author), Mans, R. (author), Pronk, J.T. (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also...

journal article 2018

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The Crabtree Effect Shapes the Saccharomyces cerevisiae Lag Phase during the Switch between Different Carbon Sources

Perez-Samper, Gemma (author), Cerulus, Bram (author), Jariani, Abbas (author), Vermeersch, Lieselotte (author), Barrajon Simancas, N. (author), Bisschops, M.M.M. (author), van den Brink, Joost (author), Solis Escalante, D. (author), Gallone, Brigida (author), De Maeyer, Dries (author), Daran-Lapujade, P.A.S. (author)

When faced with environmental changes, microbes often enter a temporary growth arrest during which they reprogram the expression of specific genes to adapt to the new conditions. A prime example of such a lag phase occurs when microbes need to switch from glucose to other, less-preferred carbon sources. Despite its industrial relevance, the...

journal article 2018

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A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9

Mans, R. (author), Wijsman, M. (author), Daran-Lapujade, P.A.S. (author), Daran, J.G. (author)

Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could...

journal article 2018

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FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae

Swiat, M.A. (author), Dashko, S. (author), den Ridder, M.J. (author), Wijsman, M. (author), van der Oost, John (author), Daran, J.G. (author), Daran-Lapujade, P.A.S. (author)

Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can mediate RNA-guided DNA cleavage at targeted...

journal article 2017