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F.J. Boonekamp
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7 records found
1
Journal article
(2026)
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Ewout Knibbe, Francine J. Boonekamp, Rachel Stuij, Philipp Savakis, Koen A.J. Pelsma, Liset Jansen, Carmen Lisset Flores, Bas Teusink, Pascale Daran-Lapujade
Protein allostery, present in all three domains of life, is key to the regulation of metabolism by allowing fast and precise control of catalysis in response to cellular demands. While metabolic pathways are frequently equipped with multiple allosterically regulated catalytic steps, experimental studies often focus on a single step, failing to capture how regulations exerted at multiple steps interact with each other for tuning pathways. Using the nearly ubiquitous Embden-Meyerhof-Parnas pathway of glycolysis as a paradigm, the present study unveils a remarkable regulatory synergy between multiple allosteric proteins of a metabolic pathway and demonstrates its impact on cell survival in dynamic environments. By using complete pathway complementation, as well as single-gene complementation, the essential regulatory steps were identified to be glucokinase, phosphofructokinase, and pyruvate kinase. Expression of these enzymes together, even in the context of the Saccharomyces cerevisiae pathway, led to imbalances in glycolysis that could only be overcome by lowering the glucokinase activity. Integrating these results with kinetic modeling and microfluidics experiments, the present work reveals the key synergistic role played by allosteric regulations in preventing glycolytic imbalance in the model eukaryote Saccharomyces cerevisiae and highlights the power of synthetic biology in addressing long-standing questions in systems biology.IMPORTANCEAll forms of life are equipped with intricate molecular mechanisms that tune their cellular responses to external and internal cues. These mechanisms are key to cells' survival in natural environments and important for the performance of bioprocesses, which are characterized by variable environments (e.g., nutrient availability). One of these molecular mechanisms, protein allostery, enables rapid fine-tuning of the rate of cellular processes by modulating protein activity in response to metabolites in vivo. Using the industrial yeast and model eukaryote Saccharomyces cerevisiae as a paradigm, the present work reveals that, in the major route for sugar utilization known as glycolysis, three distinct allosteric regulations are critical to yeast cell survival when transitioning between carbon sources. These three regulations, while not required for pathway operation per se, allow efficient and balanced pathway operation under dynamic conditions. These findings, therefore, reveal a new aspect of yeast glycolysis, one of the best-studied metabolic pathways.
...
Protein allostery, present in all three domains of life, is key to the regulation of metabolism by allowing fast and precise control of catalysis in response to cellular demands. While metabolic pathways are frequently equipped with multiple allosterically regulated catalytic steps, experimental studies often focus on a single step, failing to capture how regulations exerted at multiple steps interact with each other for tuning pathways. Using the nearly ubiquitous Embden-Meyerhof-Parnas pathway of glycolysis as a paradigm, the present study unveils a remarkable regulatory synergy between multiple allosteric proteins of a metabolic pathway and demonstrates its impact on cell survival in dynamic environments. By using complete pathway complementation, as well as single-gene complementation, the essential regulatory steps were identified to be glucokinase, phosphofructokinase, and pyruvate kinase. Expression of these enzymes together, even in the context of the Saccharomyces cerevisiae pathway, led to imbalances in glycolysis that could only be overcome by lowering the glucokinase activity. Integrating these results with kinetic modeling and microfluidics experiments, the present work reveals the key synergistic role played by allosteric regulations in preventing glycolytic imbalance in the model eukaryote Saccharomyces cerevisiae and highlights the power of synthetic biology in addressing long-standing questions in systems biology.IMPORTANCEAll forms of life are equipped with intricate molecular mechanisms that tune their cellular responses to external and internal cues. These mechanisms are key to cells' survival in natural environments and important for the performance of bioprocesses, which are characterized by variable environments (e.g., nutrient availability). One of these molecular mechanisms, protein allostery, enables rapid fine-tuning of the rate of cellular processes by modulating protein activity in response to metabolites in vivo. Using the industrial yeast and model eukaryote Saccharomyces cerevisiae as a paradigm, the present work reveals that, in the major route for sugar utilization known as glycolysis, three distinct allosteric regulations are critical to yeast cell survival when transitioning between carbon sources. These three regulations, while not required for pathway operation per se, allow efficient and balanced pathway operation under dynamic conditions. These findings, therefore, reveal a new aspect of yeast glycolysis, one of the best-studied metabolic pathways.
Journal article
(2022)
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Francine J. Boonekamp, Ewout Knibbe, Marcel A. Vieira-Lara, Melanie Wijsman, Marijke A.H. Luttik, Karen van Eunen, Maxime den Ridder, Martin Pabst, Jean Marc Daran, Pascale Daran-Lapujade
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 gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire essential pathway paves the way for establishment of species-, tissue-, and disease-specific metazoan models.
...
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 gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses. Human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, whereas hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire essential pathway paves the way for establishment of species-, tissue-, and disease-specific metazoan models.
Journal article
(2020)
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Francine J. Boonekamp, Sofiia Dashko, Jean Marc Daran, Pascale Daran-Lapujade, Donna Duiker, Thies Gehrmann, Marcel van den Broek, Maxime den Ridder, Martin Pabst, Vincent Robert, Thomas Abeel, Eline D. Postma
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 documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression, and physiology in the popular model and industrial yeast Saccharomyces cerevisiae. The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented, and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The 13 genes encoding proteins involved in the major pathway for sugar utilization (i.e., glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not affect yeast physiology and did not alter transcript abundance, protein abundance, and protein activity besides a mild effect on Gpm1. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae.
...
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 documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression, and physiology in the popular model and industrial yeast Saccharomyces cerevisiae. The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented, and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The 13 genes encoding proteins involved in the major pathway for sugar utilization (i.e., glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not affect yeast physiology and did not alter transcript abundance, protein abundance, and protein activity besides a mild effect on Gpm1. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae.
Already for millennia, microbial fermentation is used for the production of dairy products, alcoholic beverages and bread. In the last decades, the field of biotechnology has tremendously expanded and nowadays, a wide range of compounds ranging from biofuels to chemicals and pharmaceuticals is produced using microbial cell factories. The development of genetic engineering tools has greatly contributed to this rapid development. Catalysing the conversion of renewable carbohydrate feedstocks into fuels and chemicals, microbial cell factories offer a sustainable alternative to fossil resources-based production, and thereby contribute to reduce greenhouse gas emissions. The yeast Saccharomyces cerevisiae plays an important role in industrial biotechnology. Its popularity for applied research and industrial production can be attributed to several factors as its fast fermentative metabolism, its tolerance to low pH, high sugar and alcohol concentrations and its genetic tractability. S. cerevisiae possesses one of the best furbished molecular toolboxes, which makes it possible to assemble complex heterologous pathways, as was recently illustrated by the successful biosynthesis of opioids in yeast. Despite this great progress, extensive genetic remodelling of native pathways remains challenging. This can largely be explained by the high genetic redundancy present in the yeast genome, in which multiple genes encode proteins with redundant functions, and by the fact that the genes belonging to a pathway are scattered over the entire genome. The goal of this thesis was to design, set up and validate a strategy aiming at facilitating the remodelling of (essential) pathways, based on simplifying and reorganizing the yeast genome. The starting point of this research is the central carbon metabolism and in particular, as proof of concept, the glycolytic pathway.
...
Already for millennia, microbial fermentation is used for the production of dairy products, alcoholic beverages and bread. In the last decades, the field of biotechnology has tremendously expanded and nowadays, a wide range of compounds ranging from biofuels to chemicals and pharmaceuticals is produced using microbial cell factories. The development of genetic engineering tools has greatly contributed to this rapid development. Catalysing the conversion of renewable carbohydrate feedstocks into fuels and chemicals, microbial cell factories offer a sustainable alternative to fossil resources-based production, and thereby contribute to reduce greenhouse gas emissions. The yeast Saccharomyces cerevisiae plays an important role in industrial biotechnology. Its popularity for applied research and industrial production can be attributed to several factors as its fast fermentative metabolism, its tolerance to low pH, high sugar and alcohol concentrations and its genetic tractability. S. cerevisiae possesses one of the best furbished molecular toolboxes, which makes it possible to assemble complex heterologous pathways, as was recently illustrated by the successful biosynthesis of opioids in yeast. Despite this great progress, extensive genetic remodelling of native pathways remains challenging. This can largely be explained by the high genetic redundancy present in the yeast genome, in which multiple genes encode proteins with redundant functions, and by the fact that the genes belonging to a pathway are scattered over the entire genome. The goal of this thesis was to design, set up and validate a strategy aiming at facilitating the remodelling of (essential) pathways, based on simplifying and reorganizing the yeast genome. The starting point of this research is the central carbon metabolism and in particular, as proof of concept, the glycolytic pathway.
Journal article
(2018)
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Francine Boonekamp, Sofiia Dashko, Marcel van den Broek, T. Gehrmann, Jean Marc Daran, Pascale Daran-Lapujade
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 industrially relevant Saccharomyces yeast species. In this study we explore the diversity of the glycolytic and fermentative pathways within the Saccharomyces genus using S. cerevisiae, Saccharomyces kudriavzevii, and Saccharomyces eubayanus as paradigms. Sequencing data revealed a highly conserved genetic makeup of the glycolytic and fermentative pathways in the three species in terms of number of paralogous genes. Although promoter regions were less conserved between the three species as compared to coding sequences, binding sites for Rap1, Gcr1 and Abf1, main transcriptional regulators of glycolytic and fermentative genes, were highly conserved. Transcriptome profiling of these three strains grown in aerobic batch cultivation in chemically defined medium with glucose as carbon source, revealed a remarkably similar expression of the glycolytic and fermentative genes across species, and the conserved classification of genes into major and minor paralogs. Furthermore, transplantation of the promoters of major paralogs of S. kudriavzevii and S. eubayanus into S. cerevisiae demonstrated not only the transferability of these promoters, but also the similarity of their strength and response to various environmental stimuli. The relatively low homology of S. kudriavzevii and S. eubayanus promoters to their S. cerevisiae relatives makes them very attractive alternatives for strain construction in S. cerevisiae, thereby expanding the S. cerevisiae molecular toolbox.
...
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 industrially relevant Saccharomyces yeast species. In this study we explore the diversity of the glycolytic and fermentative pathways within the Saccharomyces genus using S. cerevisiae, Saccharomyces kudriavzevii, and Saccharomyces eubayanus as paradigms. Sequencing data revealed a highly conserved genetic makeup of the glycolytic and fermentative pathways in the three species in terms of number of paralogous genes. Although promoter regions were less conserved between the three species as compared to coding sequences, binding sites for Rap1, Gcr1 and Abf1, main transcriptional regulators of glycolytic and fermentative genes, were highly conserved. Transcriptome profiling of these three strains grown in aerobic batch cultivation in chemically defined medium with glucose as carbon source, revealed a remarkably similar expression of the glycolytic and fermentative genes across species, and the conserved classification of genes into major and minor paralogs. Furthermore, transplantation of the promoters of major paralogs of S. kudriavzevii and S. eubayanus into S. cerevisiae demonstrated not only the transferability of these promoters, but also the similarity of their strength and response to various environmental stimuli. The relatively low homology of S. kudriavzevii and S. eubayanus promoters to their S. cerevisiae relatives makes them very attractive alternatives for strain construction in S. cerevisiae, thereby expanding the S. cerevisiae molecular toolbox.
Pathway swapping
Toward modular engineering of essential cellular processes
Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of "pathway swapping," using yeast glycolysis as the experimental model. Construction of a "single-locus glycolysis" Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast's entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.
...
Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of "pathway swapping," using yeast glycolysis as the experimental model. Construction of a "single-locus glycolysis" Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast's entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.