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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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Using Kinetic Modelling to Infer Adaptations in Saccharomyces cerevisiae Carbohydrate Storage Metabolism to Dynamic Substrate Conditions

Lao-Martil, David (author), Verhagen, K.J.A. (author), Valdeira Caetano, Ana H. (author), Pardijs, Ilse H. (author), van Riel, Natal A.W. (author), Wahl, S.A. (author)

Microbial metabolism is strongly dependent on the environmental conditions. While these can be well controlled under laboratory conditions, large-scale bioreactors are characterized by inhomogeneities and consequently dynamic conditions for the organisms. How Saccharomyces cerevisiae response to frequent perturbations in industrial...

journal article 2023

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Expanding the genome editing toolbox of Saccharomyces cerevisiae with the endonuclease ErCas12a

Bennis, N.X. (author), Anderson, Jonah P. (author), Kok, Siebe M.C. (author), Daran, J.G. (author)

ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces...

journal article 2023

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Mimicked Mixing-Induced Heterogeneities of Industrial Bioreactors Stimulate Long-Lasting Adaption Programs in Ethanol-Producing Yeasts

Minden, Steven (author), Aniolek, Maria (author), Noorman, H.J. (author), Takors, Ralf (author)

Commercial-scale bioreactors create an unnatural environment for microbes from an evolutionary point of view. Mixing insufficiencies expose individual cells to fluctuating nutrient concentrations on a second-to-minute scale while transcriptional and translational capacities limit the microbial adaptation time from minutes to hours. This...

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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Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production

van Aalst, A.C.A. (author), de Valk, S.C. (author), van Gulik, W.M. (author), Jansen, Mickel L.A. (author), Pronk, J.T. (author), Mans, R. (author)

Product yield on carbohydrate feedstocks is a key performance indicator for industrial ethanol production with the yeast Saccharomyces cerevisiae. This paper reviews pathway engineering strategies for improving ethanol yield on glucose and/or sucrose in anaerobic cultures of this yeast by altering the ratio of ethanol production, yeast growth...

journal article 2022

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Carbon dioxide fixation via production of succinic acid from glycerol in engineered Saccharomyces cerevisiae

Malubhoy, Zahabiya (author), Bahia, Frederico Mendonça (author), de Valk, Sophie Claire (author), de Hulster, A.F. (author), Rendulić, Toni (author), Ortiz, Juan Paulo Ragas (author), Xiberras, Joeline (author), Klein, Mathias (author), Mans, R. (author), Nevoigt, Elke (author)

Background: The microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO₂). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO₂-fixation...

journal article 2022

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Predicting Metabolic Adaptation Under Dynamic Substrate Conditions Using a Resource-Dependent Kinetic Model: A Case Study Using Saccharomyces cerevisiae

Verhagen, K.J.A. (author), Eerden, S. A. (author), Sikkema, B. J. (author), Wahl, S.A. (author)

Exposed to changes in their environment, microorganisms will adapt their phenotype, including metabolism, to ensure survival. To understand the adaptation principles, resource allocation-based approaches were successfully applied to predict an optimal proteome allocation under (quasi) steady-state conditions. Nevertheless, for a general,...

journal article 2022

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Monitoring Intracellular Metabolite Dynamics in Saccharomyces cerevisiae during Industrially Relevant Famine Stimuli

Minden, Steven (author), Aniolek, Maria (author), Sarkizi Shams Hajian, Christopher (author), Teleki, Attila (author), Zerrer, Tobias (author), Delvigne, Frank (author), van Gulik, W.M. (author), Deshmukh, Amit (author), Noorman, H.J. (author), Takors, Ralf (author)

Carbon limitation is a common feeding strategy in bioprocesses to enable an efficient microbiological conversion of a substrate to a product. However, industrial settings inherently promote mixing insufficiencies, creating zones of famine conditions. Cells frequently traveling through such regions repeatedly experience substrate shortages and...

journal article 2022

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Aerobic growth physiology of Saccharomyces cerevisiae on sucrose is strain-dependent

Soares Rodrigues, C.I. (author), Wahl, S.A. (author), Gombert, Andreas K. (author)

Present knowledge on the quantitative aerobic physiology of the yeast Saccharomyces cerevisiae during growth on sucrose as sole carbon and energy source is limited to either adapted cells or to the model laboratory strain CEN.PK113-7D. To broaden our understanding of this matter and open novel opportunities for sucrose-based biotechnological...

journal article 2021

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Identification of Oxygen-Independent Pathways for Pyridine Nucleotide and Coenzyme A Synthesis in Anaerobic Fungi by Expression of Candidate Genes in Yeast

Perli, T. (author), Vos, A.M. (author), Bouwknegt, J. (author), Dekker, W.J.C. (author), Wiersma, S.J. (author), Mooiman, C. (author), Ortiz Merino, R.A. (author), Daran, J.G. (author), Pronk, J.T. (author)

Neocallimastigomycetes are unique examples of strictly anaerobic eukaryotes. This study investigates how these anaerobic fungi bypass reactions involved in synthesis of pyridine nucleotide cofactors and coenzyme A that, in canonical fungal pathways, require molecular oxygen. Analysis of Neocallimastigomycetes proteomes identified a candidate...

journal article 2021

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Directed evolution of unspecific peroxygenase in organic solvents

Martin-Diaz, Javier (author), Molina-Espeja, Patricia (author), Hofrichter, Martin (author), Hollmann, F. (author), Alcalde, Miguel (author)

Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C–H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion...

journal article 2021

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Elimination of aromatic fusel alcohols as by-products of Saccharomyces cerevisiae strains engineered for phenylpropanoid production by 2-oxo-acid decarboxylase replacement

Else-Hassing, J. (author), Buijs, Joran (author), Blankerts, Nikki (author), Luttik, M.A.H. (author), de Hulster, A.F. (author), Pronk, J.T. (author), Daran, J.G. (author)

Engineered strains of the yeast Saccharomyces cerevisiae are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use L-phenylalanine and/or L-tyrosine, generated by the yeast shikimate pathway, as aromatic...

journal article 2021

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Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Perli, T. (author), van der Vorm, Daan N.A. (author), Wassink, Mats (author), van den Broek, M.A. (author), Pronk, J.T. (author), Daran, J.G. (author)

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic...

journal article 2021

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Predicting by-product gradients of baker’s yeast production at industrial scale: A practical simulation approach

Hajian, Christopher Sarkizi Shams (author), Haringa, C. (author), Noorman, H.J. (author), Takors, Ralf (author)

Scaling up bioprocesses is one of the most crucial steps in the commercialization of bioproducts. While it is known that concentration and shear rate gradients occur at larger scales, it is often too risky, if feasible at all, to conduct validation experiments at such scales. Using computational fluid dynamics equipped with mechanistic...

journal article 2020

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Vitamin requirements and biosynthesis in Saccharomyces cerevisiae

Perli, T. (author), Wronska, A.K. (author), Ortiz Merino, R.A. (author), Pronk, J.T. (author), Daran, J.G. (author)

Chemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B-group vitamins, which are either enzyme cofactors or precursors for...

journal article 2020

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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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Squalene-Tetrahymanol Cyclase Expression Enables Sterol-Independent Growth of Saccharomyces cerevisiae

Wiersma, S.J. (author), Mooiman, C. (author), Giera, Martin (author), Pronk, J.T. (author)

Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead...

journal article 2020