Biology subjects

Daran-Lapujade, P.

Publications and source records attributed to Daran-Lapujade, P..

12 recordsLinked to original sources

bioRxiv2026

A protocol for lab-scale production of 13C yeast extract as internal standard for metabolomics and quantification of intracellular metabolites

Metabolomics enables the profiling of small-molecule metabolites and thereby captures the biochemical state of a living organism at a given moment and enables to monitor its cellular responses to stimuli. This technique has become a powerful tool in pharmaceutical research, the food industry, and microbial research. Metabolomics aims to obtain an unbiased metabolic profile; however, this is complicated by compound instability, complex and often extensive sample processing, and nonlinear responses in mass spectrometry. Therefore, correcting for metabolite loss and mass spectrometry-related artifacts is essential, typically achieved through relative quantification against an isotopically labelled internal standard for each metabolite of interest. This article describes how to produce 13C-labelled yeast extract and its use as internal standard for metabolomics. More specifically, it provides step-by-step protocols for the fed-batch fermentation, quenching, metabolite extraction, and LC-MS and GC-MS characterization of the internal standard. It also includes a protocol explaining how to use the internal standard for the quantification of metabolites in yeast samples.

biochemistry↗

bioRxiv2025

Assembly and cell-free expression of a partial genome for the synthetic cell

The de novo design and assembly of a DNA genome represents an important milestone toward the construction of a minimal synthetic cell. The genome must contain all the instructions to enable primary cellular functions, probably comprising over 150 genes with a total size over 200 kb. Here, we designed and built a partial synthetic genome that satisfies the requirements for its expression in PURE system--a minimal transcription-translation machinery reconstituted from purified elements--and its replication by the protein-primed {varphi}29 DNA replication system. The partial minimal synthetic genome (MSG) has a size of 41 kb, was assembled in yeast from 14 fragments, and harbors genes for phospholipid biosynthesis, DNA replication, and cell division. Yeast marker fragments were added between coding fragments to facilitate screening of correct assemblies and a BAC backbone was included to enable transfer from yeast to E. coli for amplification. Synthesis of all MSG-encoded proteins in PURE system was confirmed by liquid chromatography-mass spectrometry and fluorescence measurements. Moreover, we demonstrate successful compartmentalization and expression of the MSG in liposomes, as well as full-length replication of the linearized MSG by the {varphi}29 DNA replication machinery. This work provides proof-of-concept for the bottom-up assembly and cell-free expression of a functional genome for a minimal synthetic cell.

synthetic biology↗

bioRxiv2025

Exploration of mRNA-sized RNA import into Saccharomyces cerevisiae mitochondria by a combined synthetic biology and adaptive laboratory evolution approach

Efficient gene integration using RNA-guided endonucleases has not yet been achieved in the mitochondrial genome. Import of nucleic acids into mitochondria, a controversial feature, is essential for implementation of Cas9-mediated genome engineering of mitochondria. Import of short RNAs naturally occurs in mitochondria, and several putative import mechanisms and determinants have been proposed. However to date, import of gene-length RNA, required for gene integration in the mitochondrial genome, has never been described. The goal of this study was to devise and test experimental strategies to detect and improve the import of mRNA-sized RNA in mitochondria, using S. cerevisiae as model. A first fluorescence-based screening approach, relying on mitochondrial import of a fluorescent protein encoding mRNA was analyzed by fluorescence measurements, western blot and mRNA-FISH. Confounding results obtained with these different techniques made it difficult to unambiguously conclude on the occurrence of import of mRNA-sized RNAs into mitochondria. An adaptive laboratory evolution (ALE) approach, imposing a strong selection pressure for mRNA import to mitochondria, was then designed and tested to improve mitochondrial mRNA import. While the ALE approach did not improve mitochondrial mRNA import in the present study, it is a promising, unambiguous method for future studies testing different RNAs or mutants. The present study highlights remaining challenges in analytical techniques to identify RNA import to mitochondria, and introduces a novel application of ALE for studies on mitochondrial import of short and long RNA species.

synthetic biology↗

bioRxiv2023

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

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or post-transcriptional processing and splicing. Due to inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification the spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution, and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as sole carbon source, revealing the impact of growth conditions on mitochondrial RNA-expression and splicing. This study uncovered a remarkable difference in turn-over of group II introns between yeast grown in mostly fermentative and fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to explore mitochondrial transcriptome regulation and processing in a broad range of conditions relevant in human context, including aging, apoptosis and mitochondrial diseases.

microbiology↗

bioRxiv2022

Proteome dynamics during transition from exponential to stationary phase under aerobic and anaerobic conditions in yeast

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 optimising biosynthetic routes. Recent studies have demonstrated the potential of resource and proteomic allocation data in enhancing models for metabolic processes. However, comprehensive and accurate proteome dynamics data that can be used for such approaches are still very limited. Therefore, we performed a quantitative proteome dynamics study to comprehensively cover the transition from exponential to stationary phase for both aerobically and anaerobically grown yeast cells. The combination of highly controlled reactor experiments, biological replicates and standardised sample preparation procedures ensured reproducibility and accuracy. Additionally, we selected the CEN.PK lineage for our experiments because of its relevance for both fundamental and applied research. Together with the prototrophic, standard haploid strain CEN.PK113-7D, we also investigated an engineered strain with genetic minimisation of the glycolytic pathway, resulting in the quantitative assessment of over 1700 proteins across 54 proteomes. These proteins account for nearly 40% of the overall yeast proteome and approximately 99% of the total protein biomass. The anaerobic cultures showed remarkably less proteome-level changes compared to the aerobic cultures, during transition from the exponential to the stationary phase as a consequence of the lack of the diauxic shift in the absence of oxygen. These results support the notion that anaerobically growing cells lack time and resources to adapt to changes in the environment. This proteome dynamics study constitutes an important step towards better understanding of the impact of glucose exhaustion and oxygen on the complex proteome allocation process in yeast. Finally, the established proteome dynamics data provide a valuable resource for the development of resource allocation models as well as for metabolic engineering efforts.

systems biology↗

bioRxiv2022

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

The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols, particularly when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory S. cerevisiae. Sample preparation protocols may bias towards specific protein fractions or challenge the analysis of native protein modifications due to reagent-induced artefacts. Here, we performed a systematic comparison of sample preparation protocols using a matrix of different conditions commonly applied in whole cell lysate proteomics. The different protocols were evaluated for their overall fraction of identified spectra, proteome and amino acid sequence coverage, GO-term distribution and number of peptide modifications, by employing a combination of database and unrestricted modification search approaches. The best proteome and amino acid sequence coverage was achieved by using Urea combined with filter-aided or in-solution digestion protocols, where the overall outcomes were strongly influenced by the employed quenching procedure. Most importantly, the use of moderate incubation temperatures and times, circumvented excessive formation of modification artefacts. Extensive reagent-induced peptide modifications, however, were observed when using solvents such as acetone or additives such as formic acid. Moreover, several filter material-related modifications were observed when employing the filter-aided procedures. Ultimately, the best protocols enabled the identification of approximately 65-70% of all acquired fragmentation spectra, where additional de novo sequencing suggests that unidentified spectra were largely of too low spectral quality to provide confident spectrum matches. This study demonstrates the large impact of different sample preparation procedures on the proteomic analysis outcome, where the collected protocols and large sets of associated mass spectrometric raw data provide a resource to evaluate and design new protocols and guide the analysis of (native) peptide modifications in the model eukaryote yeast.

systems biology↗

bioRxiv2021

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

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 assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

bioengineering↗

bioRxiv2021

A yeast with muscle does not run faster: full humanization of the glycolytic pathway in Saccharomyces cerevisiae

While transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper the 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 both glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light novel, context-dependent responses. Remarkably, human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, while hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed the 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 to the establishment of species, tissue and disease-specific metazoan models. One Sentence SummaryThis work demonstrates the successful humanization of an entire pathway in Saccharomyces cerevisiae and establishes an attractive strategy to study (human) glycolysis architecture and regulation. HighlightsO_LIThe successful humanization of the entire glycolytic pathway in yeast offers new microbial models for both fundamental and applied studies. C_LIO_LIBoth glycolytic and moonlighting functions and turnover numbers of glycolytic enzymes are highly conserved between yeast and human. C_LIO_LIFunctionality of human hexokinases 1 and 2 in yeast requires mutations in the catalytic or allosteric binding sites. C_LIO_LICombination of single gene and full transplantation with laboratory evolution reveals context-dependent activity and evolution of glycolytic enzymes. C_LI

systems biology↗

bioRxiv2021

Top-down, knowledge-based genetic reduction of yeast central carbon metabolism

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterised 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 combinatorial metabolic engineering strategies. While isoenzymes might be important in specific environments, they could be dispensable in controlled laboratory or industrial contexts. The present study explores the extent to which the genetic complexity of the central carbon metabolism (CCM) in S. cerevisiae, here defined as the combination of glycolysis, pentose phosphate pathway, tricarboxylic acid cycle and a limited number of related pathways and reactions, can be reduced by elimination of (iso)enzymes without major negative impacts on strain physiology. Cas9-mediated, groupwise deletion of 35 from the 111 genes yielded a minimal CCM strain, which despite the elimination of 32 % of CCM-related proteins, showed only a minimal change in phenotype on glucose-containing synthetic medium in controlled bioreactor cultures relative to a congenic reference strain. Analysis under a wide range of other growth and stress conditions revealed remarkably few phenotypic changes of the reduction of genetic complexity. Still, a well-documented context-dependent role of GPD1 in osmotolerance was confirmed. The minimal CCM strain provides a model system for further research into genetic redundancy of yeast genes and a platform for strategies aimed at large-scale, combinatorial remodelling of yeast CCM.

genetics↗

bioRxiv2021

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

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 the relevant cellular constraints remain to be determined for such cells. Here we developed a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions (16304 reactions in total). The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data revealed that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation - known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimization, providing a framework to understand metabolic adaptation in eukaryal cells.

systems biology↗

bioRxiv2020

gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing

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 literature by means of Cas9 or Cas12a endonucleases and their associated gRNAs. The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native PolII or PolIII RNA polymerases. These gRNA-expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in S. cerevisiae. Using Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single DNA editing. These DNA fragments can be custom-ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR-toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other, less genetically accessible yeast species.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

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

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes Synthetic Chromosomes (SynChs) as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. SynChs made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding SynChs were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

synthetic biology↗