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[hal-05323974] Doomed by cross-feeding: How yeast supports contaminant LAB growth in modeled 2G ethanol fermentation
• Contamination by lactic acid bacteria (LAB) during fermentation is a major problem for biorefineries, resulting in yield losses of 7-22% in first-generation bioethanol with yeast viability decreasing by 83%. • LAB are known to be auxotrophous for some amino acids and vitamins (B-group). Moreover, yeast have been showed to promote LAB growth by providing essential amino acids. • Contamination by LAB was successfully simulated in 2G SScF. • Increasing yeast ratio led to quicker and stronger contamination. • Yeast provides essential amino acids to LAB contaminant in 2G fermentation improving growth and so increasing negative impact on fermentation. • Engineering yeast to limit secretion of certain amino acids, such as glutamine or aspartate, could help inhibit LAB growth and thereby reduce contamination. • A transcriptomic analysis of contaminated fermentations could provide deeper insights into yeast responses under LAB-induced stress.
ano.nymous@ccsd.cnrs.fr.invalid (Kyan Aligholi) 21 Oct 2025
https://ifp.hal.science/hal-05323974v1
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[hal-05323946] How yeast paves the way for lactic acid bacteria contamination in second generation ethanol fermentation
• Contamination by lactic acid bacteria (LAB) during fermentation is a major problem for biorefineries, resulting in yield losses of 7-22% in first-generation bioethanol production processes (Thomas et al., 2001, Narendranath et al., 1997), with yeast viability decreasing by 83% (Bayrock et al., 2004). • Key factors driving contamination in 2G biofuel context, with specific inhibitors. • Results suggest a boosting effect of yeast on the contaminant, potentially through release of essential growth factors. • Identifying metabolites fluxes between both actors through metabolomic studies will allow new contamination control strategies to disrupt this parasitic interaction.
ano.nymous@ccsd.cnrs.fr.invalid (Kyan Aligholi) 21 Oct 2025
https://ifp.hal.science/hal-05323946v1
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[hal-05323928] Innovative Solvent Production Through Genetic Engineering and Microbial consortium approaches
• Genetic engineering and cocultures are complementary approaches to improve fermentation yields • The asaccharolytic C3 producing strain Anaerotignum propionicum (Apr) was characterized in different conditions • A Δhbd mutant of Clostridium acetobutylicum (Cac) was obtained using a CRISPR-Cas9 genetic tool • Co-cultures designed to enhance propanol titer were conducted between the wild type or Δhbd mutant of Cac and Apr
ano.nymous@ccsd.cnrs.fr.invalid (Cyrielle Debeir) 21 Oct 2025
https://ifp.hal.science/hal-05323928v1
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[hal-05323908] Gene essentiality in the solventogenic Clostridium acetobutylicum DSM 792
Clostridium acetobutylicum is a solventogenic, anaerobic, gram-positive bacterium that is commonly considered the model organism for studying Acetone-Butanol-Ethanol (ABE) fermentation. The need to produce these chemicals sustainably and with a minimal impact on the environment has revived interest in research on this bacterium. The recent development of efficient genetic tools allows us to better understand the physiology of this microorganism, aiming to improve its fermentation capacities. Knowledge about gene essentiality would guide future genetic editing strategies and support the understanding of crucial cellular functions in this bacterium. In this work, we applied a transposon insertion site sequencing (TIS) method to generate large mutant libraries containing millions of independent mutants with unique insertion sites, allowing us to identify essential genes. In total, we identified a core group of 418 critical genes needed for in vitro development on rich media containing glucose as a carbon source.
ano.nymous@ccsd.cnrs.fr.invalid (Alexandre Delarouzée) 21 Oct 2025
https://ifp.hal.science/hal-05323908v1
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[hal-05323775] Filling the gaps between scales to understand biomass properties
The architecture of plant biomass is highly complex and variable depending on species and can be defined as a continuum of length-scales from molecules to particles, including polymers, nano-structures, assemblies, cells, and tissues. These scales are strongly interconnected and reflect not only chemical and structural properties of biomass but most importantly their reactivity to transformation processes such as chemical, physical, mechanical or biological reactions. In order to optimize biomass conversion (considering process selection and efficiency, cost and environmental impacts) into a range of bioproducts, detailed chemical and structural characterization is essential. However, due to natural recalcitrance of biomass, no universal markers have been highlighted able to predict biomass ability to transformation. FillingGaps project envisions to develop generic tools to not only characterize at several length scales biomass properties but also to assemble the different types of information (chemical, physical, morphological properties) acquired to propose virtual model of biomass from which specific markers could be described. Gathering 10 different French partners, high-level multiscale approaches (combining wet chemistry compositional analysis, FT-IR and fluorescence spectroscopy, photon and atomic-force microscopy, NMR, MRI, mass-spectrometry imaging, SAXS, UV and tomography synchrotron beamlines) and workflow for biomass characterization with shared platform on model biomass (maize straw, poplar and brown algae) will be applied. New molecular tools (e.g. monoclonal antibodies, CBMs, enzymes, probes) to mark and analyse biomass will be delivered. Finally, a decision-making tools will be proposed to predict biomass properties and reactivity based on easily and fast-performed analysis.
ano.nymous@ccsd.cnrs.fr.invalid (P. Assemat) 21 Oct 2025
https://hal.inrae.fr/hal-05323775v1
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[hal-05315797] An AI-driven workflow for the accelerated optimization of cell-free protein synthesis
Cell-free protein synthesis (CFPS) is a versatile tool for rapid biological prototyping. However, exploring the large number of component combinations is a very time-consuming process. Active learning (AL) is known to reduce the number of experiments required, but is rarely integrated into routine laboratory workflows. To address this, we developed a fully automated Design-Build-Test-Learn (DBTL) pipeline that streamlines this optimization process with an improved AL strategy that selects informative and diverse experimental conditions. The Design phase was created entirely using ChatGPT-4 without manual code revisions, dramatically reducing coding time. This pipeline was implemented in a modular way within the Galaxy platform, following the Findable-Accessible-Interoperable-Reusable (FAIR) principles. When applied to the optimization of colicin M and E1 in both Escherichia coli and HeLa-based CFPS systems, a 2- to 9-fold increase in yield was achieved in just four cycles. This framework enables reliable, automated workflows for routine synthetic biology.
ano.nymous@ccsd.cnrs.fr.invalid (Mostafa Khalil) 15 Oct 2025
https://hal.science/hal-05315797v1
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[hal-05281013] Microbial computing: review and perspectives
[...]
ano.nymous@ccsd.cnrs.fr.invalid (Paul Ahavi) 24 Sep 2025
https://hal.science/hal-05281013v1
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[hal-05281007] Reverse Engineering Molecules from Fingerprints through Deterministic Enumeration and Generative Models
[...]
ano.nymous@ccsd.cnrs.fr.invalid (Philippe Meyer) 24 Sep 2025
https://hal.science/hal-05281007v1
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[hal-05267743] RetroRules 2026: multi‑dataset combining chemistry and biochemistry for reaction templates
RetroRules (https://retrorules.org) is a unique compendium of reaction templates, which are generic abstractions of biochemical transformations, supporting pathway discovery, reaction prediction and enzyme engineering. The 2026 release updates biochemical sources (MetaNetX, Rhea), newly integrates chemical sources (USPTO), expands template radii to 0–10 to control specificity, adopts implicit-hydrogen and minimal atom primitive encoding for faster, more compact rules, and enables templates from mass-imbalanced reactions increasing coverage of biochemically relevant entries. Atom–atom mapping transitions to the transformer-based rxnmapper, improving mapping accuracy. The RetroRules 2026 release comprises 1,062,364 templates from 88,425 curated source reactions, with coverage across 7,391 fourth-level EC classes. A redesigned website, an updated Online Template Generator, and an OpenAPI-defined API enable multi-criteria exploration (dataset/radius/EC), deduplicated template views, and convenient exports (dataset-specific TSV/CSV/JSON bundles). Sequence annotations are refreshed from UniProt and summarized as a normalized sequence-support score to guide ranking. Together, these RetroRules evolutions provide a cross-domain resource bridging biochemistry and organic chemistry with broader coverage, controllable specificity, and better usability for high-throughput pathway design, reaction prediction and enzyme engineering.
ano.nymous@ccsd.cnrs.fr.invalid (Thomas Duigou) 18 Sep 2025
https://hal.science/hal-05267743v1
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[hal-05240051] Multi-scale characterization of the porosity in lignocellulosic biomass
[...]
ano.nymous@ccsd.cnrs.fr.invalid (Firat Goc) 04 Sep 2025
https://hal.inrae.fr/hal-05240051v1
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[hal-05240023] Chemical imaging of lignocellulosic biomass: Mapping plant chemistry
Lignocellulosic biomass (LB), which encompasses various plant samples, requires thorough characterization to optimize its use as a carbon resource. Chemical imaging simultaneously provides chemical and spatial information, offering significant benefits for LB analysis. This review presents an overview of the most advanced techniques for achieving this goal. By combining spectrometry and microscopy, microspectroscopy enables chemical imaging using various irradiation sources (IR, Raman, fluorescence, among others), allowing for the quantitative mapping of key LB components such as lignins, cellulose, and hemicelluloses. Mass Spectrometry Imaging (MSI) generates a mass spectrum for each spot of a sample thereby creating a chemical image pixel-by-pixel. MSI techniques like Matrix-Assisted Laser Desorption/Ionization (MALDI), down to 2–5 μm spatial resolution, and Secondary Ion Mass Spectrometry (SIMS), down to 300 nm for molecular analysis, effectively map small molecules in LB. In contrast, Desorption ElectroSpray Ionization (DESI) has been applied to plant extracts but remains largely unexplored for LB applications. Nuclear Magnetic Resonance (NMR) provides insight into various LB properties too. Solid-state NMR (ssNMR) and Dynamic Nuclear Polarization (DNP) help elucidate the structure of LB, sometimes aided by 3D atomistic modeling, whereas micro–Magnetic Resonance Imaging (micro-MRI) and Time-Domain (TD-NMR) probe the impact of water on LB properties.
ano.nymous@ccsd.cnrs.fr.invalid (Noah Remy) 04 Sep 2025
https://hal.inrae.fr/hal-05240023v1
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[hal-05233970] dAMN: a genome scale neural-mechanistic hybrid model to predict bacterial growth dynamics
This study presents dAMN, a hybrid neural-mechanistic model that integrates neural networks with genome-scale dynamic flux balance analysis (dFBA) to predict E. coli growth curves across diverse nutrient environments. dAMN uses neural networks to infer dynamic behavior from initial metabolite concentrations, while mechanistic constraints ensure stoichiometric and thermodynamic consistency based on the iML1515 model. The model was trained on experimental growth data from media containing various combinations of sugars, amino acids, and nucleobases, and evaluated on two test sets: one for forecasting over time and another for predicting growth dynamics on unseen media. dAMN achieved high predictive power (R² > 0.9), successfully reproducing growth and substrate depletion dynamics even without intermediate metabolite measurements. An interesting innovation of dAMN is the biologically consistent treatment of the lag phase, enabling realistic adaptation dynamics absent from standard dFBA models. dAMN stands out for its ability to generalize across combinatorial nutrient inputs and produce full growth curve predictions from minimal input data.
ano.nymous@ccsd.cnrs.fr.invalid (Jean-Loup Faulon) 02 Sep 2025
https://hal.inrae.fr/hal-05233970v2
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[hal-05231537] Machine learning in predictive biocatalysis: A comparative review of methods and applications
In recent years, machine learning has significantly advanced predictive biocatalysis, enabling innovative approaches to enzyme function prediction, biocatalyst discovery, reaction modeling, and metabolic pathway optimization. This review provides a comparative analysis of current methodologies, highlighting the intersection between computational tools and biochemical data for predictive biocatalysis applications. Key aspects covered include enzyme classification, reaction annotation, enzyme-substrate specificity, reaction outcomes, and kinetic parameter prediction. We discuss various machine learning approaches, such as neural networks with increased depth, convolutional networks, graph-based architectures, and transformer models, highlighting their respective strengths and limitations. The integration of large-scale data, representation and featurization techniques, and robust validation methods has accelerated enzyme discovery and the development of eco-friendly, sustainable biocatalytic processes. In the future, machine learning is anticipated to play a central role in connecting computational insights with practical enzyme engineering efforts, advancing applications in synthetic biology, metabolic engineering, and green biocatalysis.
ano.nymous@ccsd.cnrs.fr.invalid (Neha Tripathi) 30 Aug 2025
https://hal.science/hal-05231537v1
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[hal-05155139] A computational Framework Reveals Autofluorescence Distribution Patterns Marking Enzymatic Deconstruction of Plant Cell Wall
Plant cell wall is a renewable source of biopolymers and an alternative for fossil-based resources. However, its resistance to enzymatic deconstruction, known as recalcitrance, needs to be overcome to achieve a cost-effective transformation into bioproducts. This requires a comprehensive understanding of cell wall enzymatic deconstruction which remains under-explored at microscale, mainly due to challenges in obtaining quantitative data. We have developed a robust 4D computational framework, named HydroTrack, which comprehensively addresses the challenges to quantify cell wall deconstruction at microscale from fluorescence confocal time-lapse 3D images of cell wall enzymatic hydrolysis. HydroTrack combines divide-and-conquer strategy with temporal propagation of spatial information to effectively track extensively deconstructed cell walls. HydroTrack divides the time-lapse images into sequential clusters. The transformations are computed to register intra-cluster images. The combination of these transformations enables registration of temporally distant images to track cell wall autofluorescence intensity. To identify individual cells, HydroTrack initially segments the first 3D image of the time-lapse images. The intra-cluster transformations are then used in a propagation strategy to identify and track the individual cells during enzymatic deconstruction. Application of HydroTrack to time-lapse images of spruce wood highlighted a distinct autofluorescence intensity distribution pattern marking enzymatic hydrolysis. The quantification of cell wall volumes and cell wall accessible surface areas dynamics revealed an asynchronous impact of hydrolysis on them with volume reduction preceding the decrease in accessible surface area. HydroTrack can be easily applied to datasets from other biomass species and pretreatment types to assess the impact and efficiency of hydrolysis.
ano.nymous@ccsd.cnrs.fr.invalid (Solmaz Hossein Khani) 13 Aug 2025
https://hal.science/hal-05155139v1
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[hal-05190248] Multi-modal and multi-scale analysis of the parietal structure of plant biomass for a better understanding of its degradability
In a context where biomass valorization plays a key role in sustainable development and renewable energy production strategies, it is crucial to better understand its structure in order to identify the factors that affect its degradability. The plant cell wall, which is the main component of lignocellulosic biomass, consists of several major polymers, including cellulose, lignins, hemicelluloses and pectins. While their quantities and proportion can be determined by destructive chemical analysis, the spatial distribution of these components and their interactions at the nanometer scale remain poorly understood. Yet these are crucial factors in predicting and improving the efficiency of biomass transformation processes, particularly in the bioenergy and biobased materials sectors. In order to address these challenges, we are employing advanced imaging techniques to explore the structure of the plant wall at different scales. In this study, three complementary imagery methods are used: atomic force microscopy (AFM)1 which enables topographical and mechanical characterization at the nanometric scale, Raman spectroscopy1 which provides information on chemical composition and molecular organization, and mass spectrometry imaging2 which enables the identification and localization of specific biomolecules, in this case polysaccharides and detect local enzymatic activity. However, each technique imposes specific constraints regarding sample preparation and data treatment. Therefore, the challenge in this work is to develop an original correlative methodological approach implementing all three methods on a single object to obtain a more comprehensive and multiscale view of parietal architecture. To develop this approach, we are applying these methods to maize stem sections from two genotypes with contrasting levels of recalcitrance to degradation. This comparison will enable us to identify the structural parameters influencing biomass digestibility and optimize analysis conditions for better characterization of plant cell walls. Références : [1] Reynoud, et al. New Phytol 238 (2023) :20233-2046 [2] Leroy, et al. Bioresour Technol 353 (2022) Funding: French ANR agency, PEPR B-BEST FillingGaps project (ANR-23-PEBB-0006)
ano.nymous@ccsd.cnrs.fr.invalid (Oriane Morel) 28 Jul 2025
https://hal.inrae.fr/hal-05190248v1
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[hal-05189983] Exploration of seaweed and wheat grain by magnetic resonance imaging: Preliminary results
The Bioresources: Imaging, Biochemistry & Structure Facility (BIBS) develops and applies physical and chemical methods for the characterization of raw or transformed bioresources, at a scale ranging from molecule to object, for local, national and international projects. Applications range from the characterization of plants, organs, and biopolymers to plant derived bioresources. BIBS hosts four analytical labs, i.e. mass spectrometry, microscopy, nuclear magnetic resonance (NMR) and chemical phenotyping, and one bioinformatics lab. The NMR lab hosts a vertical wide bore 400 MHz NMR spectrometer equipped with microimaging accessory. Magnetic resonance imaging at the microscopic scale (μMRI) is one of the analytical methods used at BIBS to non-destructively explore plants and carry out in situ studies of water distribution and mobility in plants at tissue level (1) and in plant-derived bioresources (2): - in a project on seaweed as a source of polysaccharides (project PEPR B-BEST FillingGaps). Seaweed are a source of polysaccharides used in food industries and for a wide range of products including cosmetics, pharmaceuticals, bio-based chemicals and bioplastics. Two seaweeds, Saccharina latissima and Laminaria digitata, were harvested in Roscoff (France) and transported to NMR lab. They are composed of blade, stipe and holdfast. The stipe, roughly cylindrical in shape, is a stem-like structure, acting as support but, contrary to terrestrial plants, has no vascular bundles. The stipe diameter and length vary according to genotype, development stage and environment. Stipes were carefully wiped and wrapped to prevent desiccation and placed in a 20 or 30 mm tube along with a 5 mm one containing doped water for NMR signal amplitude normalization. Images were acquired at room temperature, using a multi-slice multi-echo pulse sequence in order to obtain water mobility image (T2 images) and water distribution image (normalized proton density images) and a spin-echo diffusion pulse sequence in order to obtain a water diffusion image (water Apparent-Diffusion Coefficient image). Both T2 and Diffusion images highlighted several types of tissue/structure in seaweed stipes. - in a project on the adaptation of wheat to climate change and its impact on baking quality (project FSOV Climaboul). The baking quality of the wheat grain is ‘built up’ before harvest, partly during its development on the parent plant. In the context of climate change, it is vital to develop wheat varieties that are more tolerant to environmental stresses, while maintaining their ability to be processed into bread products of stable quality. A better understanding of the effects of heat stress on grain development, composition and performance is particularly required. Wheat plants were submitted to heat stress (23/29◦C night/day) at different stages of grain development under controlled conditions in growth chambers and compared with the same cultivar grown without heat stress (15/21◦C). Individual grains were placed vertically in a homemade object holder along with a 1 mm tube containing doped water for NMR signal amplitude normalization, immediately after harvest. Images were acquired at room temperature, using a multi-slice multi-echo pulse sequence in order to obtain water mobility image (T2 images) with axial and transversal slices. T2 images highlighted water distribution in different tissues and regions of young wheat grains at the beginning of the filling stage. In this poster, we present an overview of the preliminary results of these two studies conducted at the BIBS Facility.
ano.nymous@ccsd.cnrs.fr.invalid (Catherine Deborde) 28 Jul 2025
https://hal.inrae.fr/hal-05189983v1
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[hal-04854296] Subcellular ToF-SIMS imaging of the snow algae $Sanguina\ nivaloides$ by combining high mass and high lateral resolution acquisitions
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has demonstrated great potential for metabolic imaging, yet achieving sufficient high lateral and mass resolution to reach the organelle scale still remains challenging. We have developed an approach by combining two sets of acquisition of high lateral resolution ToF-SIMS imaging (> 150 nm) and high mass resolution ToF-SIMS imaging (9,000). The data was then merged and processed using multivariate analysis (MVA), allowing for the precise identification and annotation of 85% of the main contributors to the MVA components at high lateral resolution. Insights into the electron microscopy sample preparation are provided, especially as we reveal that at least 3 different osmium-containing complexes can be found in different cellular compartments, probably due to different chemical reactions at different locations. In cells of the snow alga Sanguina nivaloides, living in a natural environment limited in nutrients such as phosphorus (P), we were able to map elements and molecules within their subcellular context, allowing for the identification of organelles at a resolution of 100 nm as confirmed by correlative electron microscopy. It was thus possible to highlight that S. nivaloides likely absorbed selectively some inorganic P forms, provided by P-rich dusts deposited on the snow surface. S. nivaloides cells could maintain phosphorylations in the stroma of the chloroplast, consistently with the preservation of photosynthesis activity. The presented method can thus overcome the current limitations of ToF-SIMS for subcellular imaging and contribute to the understanding of key questions such as P homeostasis and other cell physiological processes.
ano.nymous@ccsd.cnrs.fr.invalid (Claire Seydoux) 25 Jul 2025
https://hal.science/hal-04854296v1
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[hal-05170246] Cell-free genome-wide transcriptomics through machine learning optimization
Despite advances in transcriptomics, understanding of genome regulation remains limited by the complex interactions within living cells. To address this, we performed cell-free transcriptomics by developing a platform using an active learning workflow to explore over 1,000,000 buffer conditions. This enabled us to identify a buffer that increased mRNA yield by 20-fold, enabling cell-free transcriptomics. By employing increasingly complex conditions, our approach untangles the regulatory layers controlling genome expression. Main<p>Lysate-based cell-free systems (CFS) are valuable platform for studying the living and elucidating molecular mechanisms 1-3 . Transcription and translation are restored in bacterial lysates by supplementing with a buffer containing ATP regeneration substrates, crowding agents, salts, and essential precursors such as nucleotides and amino acids. As such, CFS are simpler than living cells in terms of molecular interactions as they eliminate complex and interfering biological processes such as cell division and membrane-related functions. For example, previous experiments showed that production and metabolic burdens can be untangled using cell-free compared with in vivo measurements 4 . CFS provide an open platform ideally suited for protein production, highthroughput prototyping of genetic circuits and parts, biosensing biomanufacturing and gene expression analysis and modelling 5-7 . Genome expression in living cells is routinely analyzed using RNA-seq 8 ; however, low transcript yields in CFS have so far limited the application of this technique in vitro 9 . Here, we developed a general methodological pipeline that integrates active learning loops with high-throughput experimentation to optimize mRNA production in Escherichia coli BL21(DE3) CFS. This approach enabled transcript abundances sufficient for RNA-seq-based, genome-wide transcriptomic analysis. As proof of concept, we applied the pipeline to enhance RNA synthesis by T7 RNA polymerase (T7 RNAP) and performed in vitro transcriptomic profiling of the phage T7 genome.
ano.nymous@ccsd.cnrs.fr.invalid (Léa Wagner) 18 Jul 2025
https://hal.science/hal-05170246v1
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[hal-05155548] Cell-free genome-wide transcriptomics through machine learning optimization
Despite advances in transcriptomics, understanding of genome regulation remains limited by the complex interactions within living cells. To address this, we performed cell-free transcriptomics by developing a platform using an active learning workflow to explore over 1,000,000 buffer conditions. This enabled us to identify a buffer that increased mRNA yield by 20-fold, enabling cell-free transcriptomics. By employing increasingly complex conditions, our approach untangles the regulatory layers controlling genome expression.
ano.nymous@ccsd.cnrs.fr.invalid (Léa Wagner) 10 Jul 2025
https://hal.inrae.fr/hal-05155548v1
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[hal-05147856] Functional study of Phaeodactylum tricornutum Seipin highlights specificities of lipid droplets biogenesis in diatoms
Summary Diatoms are a major phylum of microalgae, playing crucial ecological roles. They derive from secondary endosymbiosis, leading to a complex intracellular architecture. Their ability to store oil in lipid droplets (LDs) upon unfavourable conditions has raised interest for applications, in particular biofuels, yet Lipid Droplet (LD) biogenesis mechanisms in these organisms remain poorly understood. Here, we functionally characterize the homolog of Seipin, a major actor of LD biogenesis, in Phaeodactylum tricornutum. We used an in silico approach to analyze the evolutionary origin of PtSeipin and its specific features. Then, we used a functional genetics approach with a combination of confocal and electronic microscopy and lipidomics to characterize the protein function. We provide evidence that Stramenopiles Seipins were inherited from the host during secondary endosymbiosis. The localization of PtSeipin highlights participation of the plastid's most external membrane in LD biogenesis. Finally, the knock‐out (KO) of PtSeipin leads to a strong increase of triacylglycerol (TAG) accumulation, a feature that had not been observed in adipogenic or oleaginous cells and is greatly enhanced following high light exposure. Our results suggest a redirection of lipid fluxes toward TAG synthesis, reduced TAG recycling or a combination of both in PtSeipin KO.
ano.nymous@ccsd.cnrs.fr.invalid (Damien Le Moigne) 07 Jul 2025
https://hal.inrae.fr/hal-05147856v1
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[hal-05134741] An M13 phagemid toolbox for engineering tuneable DNA communication in bacterial consortia
Intercellular communication is essential for distributed genetic circuits operating across cells in multicellular consortia. While diverse signalling molecules have been employed--ranging from quorum sensing signals, secondary metabolites, and pheromones to peptides, and nucleic acids--phage-packaged DNA offers a highly programmable method for communicating information between cells. Here, we present a library of five M13 phagemid variants with distinct replication origins, including those based on the Standard European Vector Architecture (SEVA) family, designed to tune the growth and secretion dynamics of sender strains. We systematically characterize how intracellular phagemid copy number varies with cellular growth physiology and how this, in turn, affects phage secretion rates. In co-cultures, these dynamics influence resource competition and modulate communication outcomes between sender and receiver cells. Leveraging the intercellular CRISPR interference (i-CRISPRi) system, we quantify phagemid transfer frequencies and identify rapid-transfer variants that enable efficient, low-burden communication. The phagemid toolbox developed here expands the repertoire of available phagemids for DNA-payload delivery applications and for implementing intercellular communication in multicellular circuits.
ano.nymous@ccsd.cnrs.fr.invalid (Abhinav Pujar) 04 Jul 2025
https://hal.science/hal-05134741v1
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[hal-05119566] Genotype-dependent response to water deficit: increases in maize cell wall digestibility occurs through reducing both p-coumaric acid and lignification of the rind
Introduction: The compositional dynamics of the cell wall are influenced by drought, and it has been demonstrated that water deficit induces significant changes in its main components. Moreover, changes in cell wall concentration and distribution in response to water deficit affect maize degradability. Material and methods: This study presents a histological and biochemical analysis of thirteen maize inbred lines, evaluated over two years in Pobra de Brollón (Spain) and Mauguio (France) under contrasting water availability conditions. Our aim was to investigate the environmental and genotypic impacts on histological and biochemical profiles, to assess in vitro cell wall digestibility under water deficit, and to explore how these responses relate to changes in cell wall composition and structure. Results and discussion: Overall, we observed greater concentrations of p-coumaric acid under control conditions, with significant decreases in stressed conditions at each location. Histologically, we found an increase in non-lignified tissues under water deficit conditions across all tissues at each location as well. In terms of in vitro cell wall digestibility (IVCWRD), significant increases were detected in response to water deficit. Additionally, genotype-dependent response patterns were evident, revealing two distinct behavioural groups. Notably, in plastic genotypes, increases in IVCWRD in response to water deficit were concomitant to reductions in p-coumaric acid content and a decrease in red-stained lignified tissues in the rind. This study emphasizes the complex, genotype-dependent responses to water deficit, underscoring the important roles of plasticity and stability in shaping the impact on maize cell wall digestibility; paving the way to breed for adapted genotypes to face climate changes.
ano.nymous@ccsd.cnrs.fr.invalid (Ana López-Malvar) 18 Jun 2025
https://hal.inrae.fr/hal-05119566v1
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[hal-04964370] Light, CO<sub>2</sub>, and carbon storage in microalgae
Microalgae exhibit remarkable adaptability to environmental changes by integrating light and CO2 signals into regulatory networks that govern energy conversion, carbon fixation, and storage. Light serves not only as an energy source for photosynthesis but also as a regulatory signal mediated by photoreceptors. Specific light spectra distinctly influence carbon allocation, driving lipid or starch biosynthesis by altering transcriptional and metabolic pathways. The ratio of ATP to NADPH imbalances significantly impact carbon allocation toward lipid or starch production. To maintain this balance, alternative electron flow pathways play critical roles, while inter-organelle redox exchanges regulate cellular energy states to support efficient carbon storage. The CO2-concentrating mechanism (CCM) enhances photosynthetic efficiency by concentrating CO2 at Rubisco, energized by ATP from photosynthetic electron transport. This review examines how light receptors, energy-producing pathways, and the CCM interact to regulate carbon metabolism in microalgae, emphasizing their collective roles in balancing energy supply and carbon storage.
ano.nymous@ccsd.cnrs.fr.invalid (Yasuyo Yamaoka) 18 Jun 2025
https://hal.science/hal-04964370v1
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[hal-05106860] Greener catalytic oxidation of furan derivatives with the help of new catalytic systems made through a bottom-up approach
Introduction To reduce our dependence on fossil resources and promote sustainability, biomass is a key carbon source. Carbohydrate-rich biomass yields a variety of valuable molecules, including 2,5-furandicarboxylic acid (FDCA) and 2,5-diformylfuran (DFF) used as monomers or for pharmaceutical syntheses, which can be produced through the catalytic oxidation of 5-hydroxymethylfurfural (5-HMF). Since 2010, many studies have investigated the selective oxidation of 5-HMF, primarily using noble metalsupported catalysts (Au, Pd, Pt, Ru) in the presence of an excess of soluble base(1). However, the high cost of these metals, combined with wastewater treatment issues, highlight the need for new approaches. In this context, a bottom-up strategy appears promising. “Quasi-homogeneous” catalysis by stable metallic colloids in solutions can be an effective means of optimizing the reaction parameters towards greener processes. Moreover, this method has been relatively underexplored for 5-HMF oxidation. Few systems, such as core-shell Pt-Fe3O4 nanoparticles, have been applied to FDCA formation(2), while NH4.V3O8-Fe3O4 nanoparticles have been used for DFF(3). In the context of a project funded by PEPR BBEST-FURFUN (2023-2026) focused on bio-sourced furans valorizations, we will present in this communication our strategy for developing an “ideal” catalytic system following a bottom-up approach: discovering a stable and efficient heterogeneous catalysts from a "quasi-homogeneous" colloidal phase able to work in the absence of soluble bases (Figure 1). We will detail our work from the synthesis of stable metal colloids in aqueous media to support palladium nanoparticles on carriers for the selective oxidation of HMF into DFF or FDCA under mild reaction conditions. Materials &amp; Methods PVP-stabilized palladium colloids (Pd NPs) were prepared through a reliable method(4). Na₂PdCl₄ and PVP (10 eq.) were separately dissolved in water, combined, and stirred vigorously for 0.5 hours. Pd NPs were then formed by reduction with NaBH₄ (2.5 eq.) at room temperature stirring for 1 day. The Pd NPs were characterized by ICP and TEM, and the colloidal solution has shown to be stable over weeks. For catalytic experiments, 40 mL of a 0.5 wt% HMF solution in water, Na₂CO₃ (≤1.2 eq./HMF) and palladium colloidal solution (0.55 mol.%), were placed in a three-neck flask, exposed to a flow of wet air, and stirred at 1000 rpm at 25 or 80°C for 48 hours. Reaction mixtures were analyzed via HPLC. Results &amp; Discussion We previously reported the use of palladium based catalysts for the catalytic oxidation of carbonyl compounds4, and we present here our initial results on “quasi-homogeneous” catalysis using palladium colloids. The results observed will serve as a baseline for further development of catalysts based on non-critical metals like Cu, Ag or Au. First studies focused on particle size and stability of Pd NPs aqueous solutions, as well as the effect of the palladium colloids on the activity and selectivity of 5-HMF oxidation. Parameters like base concentration, temperature, and air/O₂ flow rate were evaluated to clarify their roles in the reaction pathways, comparing our results to those of existing literature. TEM analyses revealed uniform particles size of around 3.5 nm with a narrow distribution showing also that, colloidal solutions are stable for at least one month. As an example of catalytic conditions variations, under air bubbling, the temperature of the reaction affects the selectivity. While at 25°C, HMF conversion reaches 60% with 80-90% selectivity to HMFCA, at 80°C, HMF is fully converted with a selectivity up to 70% to FDCA. BIBLIOGRAPHY (1). Zhang, Z., Deng, K., ACS Catal., 2015, 5, 11 (2). Zhang, Y., Xue, Z., Wang, J., Zhao, X., Deng, Y., Zhao, W., Mu, T., RSC Adv., 2016, 6, 56 (3). Lai, J., Zhou, S., Cheng, F., Guo, F., Liu, X., Xu, Q., Yin, D., Catal. Lett., 2020, 150, 1301 (4). Bourbiaux, D., Mangematin, S., Djakovitch, L., Rataboul, F., Catal. Lett., 2021, 151, 3239
ano.nymous@ccsd.cnrs.fr.invalid (Lénaïck Hervé) 11 Jun 2025
https://hal.science/hal-05106860v1
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[hal-05106159] A bottom-up approach to design new catalytic systems for the mild oxidation of furan derivatives
Introduction To reduce our dependence on fossil resources and promote sustainability, biomass is a key carbon source. Carbohydrate-rich biomass yields a variety of valuable molecules, including 2,5- furandicarboxylic acid (FDCA) and 2,5-diformylfuran (DFF), which can be produced through the catalytic oxidation of 5-hydroxymethylfurfural (5-HMF) for applications as monomers or pharmaceuticals. Since 2010, many studies have investigated the selective oxidation of 5-HMF, primarily using noble metal-supported catalysts (Au, Pd, Pt, Ru) and several equivalents of soluble base. 1 However, the high cost of these metals, combined with wastewater treatment issues, highlight the need for new approaches. In this context, a bottom-up strategy appears promising. “Quasi-homogeneous” catalysis through metallic colloids stable in solutions may provide an effective way to identify the most suitable metal(s) for the catalytic system. This method has been relatively underexplored for 5-HMF oxidation. Some systems, such as core-shell Pt-Fe3O4 nanoparticles, have been applied to FDCA formation2 , while NH4.V3O8-Fe3O4 nanoparticles have been used for DFF. 3 In the context of a project funded by PEPR BBEST-FURFUN (2023-2026) focused on bio-sourced furan valorization, we will present in this communication our strategy for developing an “ideal” catalytic system through a bottom-up approach: this is the creation of a "quasi-homogeneous" colloidal phase and then the progression to stable and efficient heterogeneous catalysts (Figure 1). We will detail here our developments for the synthesis of stable palladium colloids in aqueous media for the selective oxidation of HMF to either DFF or FDCA under mild conditions. Materials &amp; Methods We prepared PVP-stabilized palladium colloids (Pd NPs) through a reliable method.4 Na₂PdCl₄ and PVP (10 eq.) were separately dissolved in water, combined, and stirred vigorously for 0.5 hours. Pd NPs were then formed by reduction with NaBH₄ (2.5 eq.) at room temperature and stirring for 1 day. The Pd NPs were characterized by ICP and TEM, and the colloidal solution has shown to be stable over weeks. For catalytic experiments, 40 mL of a 0.5 wt% HMF solution in water, Na₂CO₃ (≤1.2 eq./HMF) and palladium colloidal solution (0.55 mol.%), were placed in a three-neck flask, exposed to a flow of wet air, and stirred at 1000 rpm at 25 or 80°C for 48 hours. Reaction mixtures were analyzed via HPLC. Results &amp; Discussion Palladium is renowned for the catalytic oxidation of carbonyl compounds, and we present initial results on “quasi-homogeneous” catalysis using palladium colloids, that will serve as a baseline for comparison with non-critical metals (e.g., Cu, Ag, Au). First analysis focused on particle size and stability of Pd NP solutions in aqueous media, as well as the effect of palladium colloids on the activity and selectivity for 5-HMF oxidation. Various catalytic tests examined parameters like base concentration, temperature, and air/O₂ flow rate to clarify their roles in the reaction pathway, with the support of existing literature. TEM analysis revealed uniform particles of around 3.5 nm with a narrow size distribution and stable, as colloidal solution for at least one month. As an example of catalytic conditions variations, temperature effect showed that at 25°C, HMF conversion reached 70% with 80% selectivity into HMFCA, while at 80°C, HMF was fully converted with 100% selectivity into FDCA. Significance This study participates to the advances towards the implementation of robust routes for the very important reaction of oxidation of 5-HMF into DFF or FDCA, particularly by using more eco-friendly procedures limiting the use of oxygen pressure, as well as expensive reagents and solvents. References 1. Z. Zhang, K. Deng, ACS Catal., 2015, 5, 11. 2. Y. Zhang, Z. Xue, J. Wang, X. Zhao, Y. Deng, W. Zhao, T. Mu, RSC Adv., 2016, 6, 56. 3. J. Lai, S. Zhou, F. Cheng, D. Guo, X. Liu, Q. Xu, D. Yin, Catal. Lett., 2020, 150, 1301. 4. D. Bourbiaux, S. Mangematin, L. Djakovitch, F. Rataboul, Catal. Lett., 2021, 151, 3239
ano.nymous@ccsd.cnrs.fr.invalid (Lénaïck Hervé) 10 Jun 2025
https://hal.science/hal-05106159v1
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[hal-05076578] Multivariate analysis of IR spectroscopic isotherms: a new method for the characterization of adsorption at the molecular scale
In this communication we show how in situ IR spectroscopy combined with multivariate analysis can be used to identify and characterize quantitatively the adsorption, in particular alcohol on zeolites.
ano.nymous@ccsd.cnrs.fr.invalid (Reda Aboulayt) 21 May 2025
https://hal.science/hal-05076578v1
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[hal-05035782] A distinct autofluorescence distribution pattern marks enzymatic deconstruction of plant cell wall
Achieving an economically viable transformation of plant cell walls into bioproducts requires a comprehensive understanding of enzymatic deconstruction. Microscale quantitative analysis offers a relevant approach to enhance our understanding of cell wall hydrolysis, but becomes challenging under high deconstruction conditions. This study comprehensively addresses the challenges of quantifying the impact of extensive enzymatic deconstruction on plant cell wall at microscale. Investigation of highly deconstructed spruce wood provided spatial profiles of cell walls during hydrolysis with remarkable precision. A distinct cell wall autofluorescence distribution pattern marking enzymatic hydrolysis along with an asynchronous impact of hydrolysis on cell wall structure, with cell wall volume reduction preceding cell wall accessible surface area decrease, were revealed. This study provides novel insights into enzymatic deconstruction of cell wall at under-investigated cell scale, and a robust computational pipeline applicable to diverse biomass species and pretreatment types for assessing hydrolysis impact and efficiency.
ano.nymous@ccsd.cnrs.fr.invalid (Solmaz Hossein Khani) 16 Apr 2025
https://hal.science/hal-05035782v1
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[hal-05027262] Reconstituting alternative life using the test-bed of cell-free systems
The current form of life on Earth is the outcome of a series of biochemical events that led to the formation of early life with a specific molecular composition encoding its many functions. However, other trajectories of events with widely different outcomes could have led to alternative forms of life with different compositions. Although these "roads not taken" have been hypothesised in Origins of Life research for long, only recently have advances in several life technologies enabled us to experimentally explore them. Here we discuss how one such technology, cell-free expression systems (CFS), offers a promising avenue for reconstitution of specific biological functions in a controlled in vitro environment. Breaking free of the complex biochemical interactions of the closed cellular compartment facilitates testing of engineered alternatives of these functions, in whole or part, reconstituted using both natural and synthetic orthogonal components. In this perspective, we focus on how CFS has enabled characterisation and reconstitution of several steps of biological information transfer using alternative machinery (XNAs, RNAPs, ribosomes, non-canonical amino acids, tRNA synthetases, mirror-image biomolecules), including in alternative biochemistries and compartments, identifying the applicable constraints. Once these alternative functions have been characterised and co-optimised, how do we foresee their reintegration into alternative life forms of the future?
ano.nymous@ccsd.cnrs.fr.invalid (Maud Hofmann) 09 Apr 2025
https://hal.inrae.fr/hal-05027262v1
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[hal-04463739] Phototropin connects blue light perception to starch metabolism in green algae
In photosynthetic organisms light acts as an environmental signal to control their development and physiology, and as energy source to drive the conversion of CO 2 into carbohydrates used for growth or storage. The main storage carbohydrate in green algae is starch, which accumulates during the day and is broken down at night to meet cellular energy demands. The signalling role of light quality in the regulation of starch accumulation remains unexplored. Here, we report that in the model green alga Chlamydomonas reinhardtii blue light perceived by the photoreceptor PHOTOTROPIN causes dephosphorylation of the PHOTOTROPIN-MEDIATED SIGNALLING KINASE 1 that then suppresses starch accumulation by inhibiting the expression of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE. Our results provide an in-depth view of how photoreceptor-mediated signalling controls microalgal carbon metabolism. One-Sentence Summary Blue light perception by PHOTOTROPIN triggers kinase-mediated signaling to inhibit starch accumulation in the green alga Chlamydomonas .
ano.nymous@ccsd.cnrs.fr.invalid (Yizhong Yuan) 17 Mar 2025
https://hal.science/hal-04463739v1
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[hal-04984275] Biodegradability of tomato stem-reinforced composites: Towards a virtuous approach to local and circular waste upcycling
The current method of producing tomatoes in greenhouses uses petro-sourced plastic accessories that contaminate plant waste when the greenhouses are emptied. For this reason, this study aims to develop a biodegradable material to replace plastic accessories. To evaluate the feasibility of using tomato byproduct as reinforcements in a range of biobased and biodegradable thermoplastic materials, the compound degradability was investigated though biochemical and imaging approaches. The first set of experiments carried out on the tomato stem showed that the enzymatic degradation by a mixture of cellulases and pectinases efficiently removed constitutive biopolymers, and that the average size and the polydispersity decreased during treatment. The largest particles became more irregular, highlighting the enzyme-recalcitrant domains. When compounded with different matrix polymers (PBS, PBAT/PHA or PBAT/PLA), tomato stem particles remained susceptible to enzymatic degradation. Tomography analysis showed that all the degraded samples exhibited a large increase in porosity, the largest increase being observed in the PLA-containing specimens. This fully circular approach from waste to useful compounds for horticulture and market gardening is a promising way of upcycling tomato biomass, compatible with end-of-life composting.
ano.nymous@ccsd.cnrs.fr.invalid (Estelle Bonnin) 12 Mar 2025
https://hal.science/hal-04984275v1
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[hal-04926148] Converting biowaste into biomolecules using saline and non-saline microbial electrolyzers
Microbial electrolysis cells (MECs) are promising technologies for the production of multi-carbon compounds by electrotrophic microorganisms at the cathode, using energy and carbon extracted from organic waste by electrogenic microorganisms at the anode. The nature of the products formed depends on the taxonomic composition of the microbial community. This study examines the impact of electrolyte salinity on the microbial assembly of electroactive consortia. Salinity is a key performance parameter in bioelectrochemical systems, as it helps reduce internal resistance and optimize energy efficiency. The study compares hypersaline reactors developed from inocula collected from salt marshes with a non-saline reference system using sludge from an industrial digester (Desmond-Le Quéméner et al., 2019). Both types of reactors are operated in triplicates, each combining a bioanode with a homoacetogenic biocathode obtained by heat treatment of the inocula to eliminate methanogenic archaea. An intermediate compartment, separated by ion-exchange membranes, is integrated to extract and concentrate carboxylates formed at the cathode via electrodialysis. Different electrode configurations are used: a flat anode to maximize electrocatalytic activity and a granular cathode to increase the exchange surface between electrons and the microbial community. These design elements allow for the selection and adaptation of specific communities in each compartment. Initial tests with different types of saline electrolytes revealed the impact of salt composition on the catalytic performance of microbial electrolysis reactors. Reactors operated with NaCl-based salt solutions showed promising results in terms of energy efficiency, with high current density measurements. The production of carboxylates at the cathode by both types of microbial communities was demonstrated, providing a technological proof of concept for the proper functioning of the electrolysis cells. Ribotag sequencing analyses of 16S rDNA and rRNA are used to describe the adaptation process of microbial consortia to specific electrode configurations and the structuring of taxonomic and functional assemblages from the same initial community under saline and non-saline conditions. Reference: Desmond-Le Quéméner, E., Bridier, A., Tian, J.-H., Madigou, C., Bureau, C., Qi, Y., &amp; Bouchez, T. (2019). Biorefinery for heterogeneous organic waste using microbial electrochemical technology. Bioresource Technology, 292, 121943. https://doi.org/10.1016/j.biortech.2019.121943
ano.nymous@ccsd.cnrs.fr.invalid (Louise Rigaud) 03 Feb 2025
https://hal.inrae.fr/hal-04926148v1
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[hal-04738744] The DYRKP1 kinase regulates cell wall degradation in Chlamydomonas by inducing matrix metalloproteinase expression
The cell wall of plants and algae is an important cell structure that protects cells from changes in the external physical and chemical environment. This extracellular matrix, composed of polysaccharides and glycoproteins, must be constantly remodeled throughout the life cycle. However, compared to matrix polysaccharides, little is known about the mechanisms regulating the formation and degradation of matrix glycoproteins. We report here that a plant kinase belonging to the DUAL-SPECIFICITY TYROSINE PHOSPHORYLATION-REGULATED KINASE (DYRK) family present in all eukaryotes regulates cell wall degradation after mitosis of Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases (MMPs). Without the plant DYRK kinase (DYRKP1), daughter cells cannot disassemble parental cell walls and remain trapped inside for more than 10 days. On the other hand, the DYRKP1 complementation line shows normal degradation of the parental cell wall. Transcriptomic and proteomic analyses indicate a marked down-regulation of MMP gene expression and accumulation, respectively, in the dyrkp1 mutants. The mutants deficient in MMPs retain palmelloid structures for a longer time than the background strain, like dyrkp1 mutants. Our findings show that DYRKP1, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides insight into the life cycle regulation in plants and algae.
ano.nymous@ccsd.cnrs.fr.invalid (Minjae Kim) 25 Jan 2025
https://hal.science/hal-04738744v1
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[hal-04878044] Machine learning thermodynamic perturbation theory offers accurate activation free energies at the RPA level for alkene isomerization in zeolites
The determination of accurate free energy barriers for reactions catalyzed by proton-exchanged zeolites by quantum chemistry approaches is a challenge. While ab initio molecular dynamics is often required to sample correctly the various states described by the system, the level of theory also has a crucial impact. In the present work, we report the determination of accurate barriers for a type B isomerization of a monobranched C7 alkene (4-methyl-hex-1-ene) into a dibranched tertiary cation inside a protonated chabazite zeolite. This is done by using the Machine Learning Thermodynamic Perturbation Theory (MLPT) at the Random Phase Approximation (RPA) level, on the basis of blue-moon sampling dynamic data obtained at the Generalized Gradient Approximation (GGA) level (PBE+D2). The comparison of PBE+D2 and RPA profiles shows that the former overstabilizes cationic intermediates with respect to neutral ones. The transition state of the isomerization is a non-classical edge protonated cyclopropane, the stabilization of which is lower than that of the π-complex when PBE+D2 is replaced by RPA, but higher than that of the classical tertiary carbenium. Consequently, the backward isomerization barrier is decreased. Applying the MLPT approach to recompute the free energy barriers with various dispersion correction schemes to the PBE energies shows that none of the schemes is sufficient to improve both the forward and backward barriers with respect to the RPA reference. These data complement previously determined alkene cracking barriers [Rey et al., Angew. Chem., Int. Ed., 2024, 63, e202312392], thanks to which it is possible to compare the presently determined barriers with reference experimental data [Schweitzer et al., ACS Catal., 2022, 12, 1068–1081]. The agreement with experiments is significantly improved at the RPA with respect to GGA. Chemical accuracy is approached (maximum deviation of 6.4 kJ mol−1), opening the door to predictive kinetic modelling starting from first principles approaches.
ano.nymous@ccsd.cnrs.fr.invalid (Jérôme Rey) 09 Jan 2025
https://ifp.hal.science/hal-04878044v1
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[hal-04857279] Phage-mediated intercellular CRISPRi for biocomputation in bacterial consortia
Coordinated actions of cells in microbial communities and multicellular organisms enable them to perform complex tasks otherwise difficult for single cells. This has inspired biological engineers to build cellular consortia for larger circuits with improved functionalities while implementing communication sy stems f or coordination among cells. Here, w e in v estigate the signalling dynamics of a phage-mediated synthetic DNA messaging system and couple it with CRISPR interference to build distributed circuits that perform logic gate operations in multicellular bacterial consortia. We find that growth phases of both sender and receiver cells, as well as resource competition between them, shape communication outcomes. L e v eraging the easy programmability of DNA messages, we build eight orthogonal signals and demonstrate that intercellular CRISPRi (i-CRISPRi) regulates gene expression across cells. Finally, we multiplex the i-CRISPRi system to implement se v eral multicellular logic gates that in v olv e up to se v en cells and tak e up to three inputs simultaneously, with single-and dual-rail encoding: NOT, YES, AND and AND-AND-NOT. The communication system developed here lays the groundwork for implementing complex biological circuits in engineered bacterial communities, using phage signals for communication.
ano.nymous@ccsd.cnrs.fr.invalid (Abhinav Pujar) 28 Dec 2024
https://hal.science/hal-04857279v1
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[hal-04731720] Monogalactosyldiacylglycerol synthase isoforms play diverse roles inside and outside the diatom plastid
Diatoms derive from a secondary endosymbiosis event, which occurred when a eukaryotic host cell engulfed a red alga. This led to the formation of a complex plastid enclosed by four membranes: two innermost membranes originating from the red alga chloroplast envelope, and two additional peri- and epiplastidial membranes (PPM, EpM). The EpM is linked to the endoplasmic reticulum (ER). The most abundant membrane lipid in diatoms is monogalactosyldiacylglycerol (MGDG), synthesized by galactosyltransferases called MGDG synthases (MGDs), conserved in photosynthetic eukaryotes and considered to be specific to chloroplast membranes. Similar to angiosperms, a multigenic family of MGDs has evolved in diatoms, but through an independent process. We characterized MGDα, MGDβ and MGDγ in Phaeodactylum tricornutum, combining molecular analyses, heterologous expression in Saccharomyces cerevisiae, and studying overexpressing and CRISPR-Cas9-edited lines. MGDα localizes mainly to thylakoids, MGDβ to the PPM, and MGDγ to the ER and EpM. MGDs have distinct specificities for diacylglycerol, consistent with their localization. Results suggest that MGDα is required for thylakoid expansion under optimal conditions, while MGDβ and MGDγ play roles in plastid and non-plastid membranes and in response to environmental stress. Functional compensation among MGDs likely contributes to diatom resilience under adverse conditions and to their ecological success.
ano.nymous@ccsd.cnrs.fr.invalid (Nolwenn Guéguen) 03 Dec 2024
https://hal.science/hal-04731720v2
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[hal-04715274] Harnessing Lignocellulolytic and Electrogenic Potential: Insights from Shewanella oneidensis MR-1 and Cellulomonas Strains on Lignocellulosic Biomass
The biodegradable and renewable nature of lignocellulosic biomass (LCB) has gained significant interest in recent years. This study explores the lignocellulolytic and electrogenic potential of Shewanella oneidensis MR-1, Cellulomonas fimi ATCC 484, and Cellulomonas biazotea NBRC 12680 on LCB. Two strategies were tested: assessing strains LCB degradation ability under non-electrochemical and electrochemical conditions. Strain selection was based on literature, and bioinformatical analyses were conducted to predict CAZymes and carbohydrate degradation pathways. Cellulomonas strains have a potential to degrade LCB due to high CAZyme count and specific metabolic pathways. Strains growth capacity on LCB was evaluated by culturing without electrodes on LCB for 12 days, showing superior growth on wheat bran compared to wheat straw. Enzymatic assays indicate laccase activity in all strains, highest in C. biazotea NBRC 12680 (11.66 IU). The strains ability to form electrogenic biofilms on carbon cloth anodes polarized at +0.2 V (vs Ag/AgCl) was evaluated. The results indicate that bioanodes can function with wheat bran (max current density: 14.92 mA m −2 ), with voltammograms showing redox activities. Attenuated total reflection Fourier transform infrared spectroscopy shows lignin and protein degradation in both electrochemical and non-electrochemical experiments. These findings suggest potential use of these strains in electro-microbial systems with LCB.
ano.nymous@ccsd.cnrs.fr.invalid (Animut Assefa Molla) 30 Sep 2024
https://hal.science/hal-04715274v1
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[hal-04703221] Knocking out the carboxyltransferase interactor 1 (CTI1) in Chlamydomonas boosted oil content by fivefold without affecting cell growth
ABSTRACT The first step in chloroplast de novo fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACCase). As the rate-limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homolog of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein, interacts with the Chlamydomonas α-carboxyltransferase (Crα-CT) subunit of the ACCase by yeast two-hybrid protein-protein interaction assay. Three independent CRISPR-Cas9 mediated knock-out mutants for CrCTI1 each produced an “enhanced oil” phenotype, accumulating 25% more total fatty acids and storing up to five-fold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Crα-CT. However, mutating all six predicted phosphorylation sites within Crα-CT to create a phosphomimetic mutant reduced significantly this pairwise interaction. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid β-oxidation pathways, to enable cells adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for genetic engineering of microalgae for biotechnology purposes.
ano.nymous@ccsd.cnrs.fr.invalid (Zhongze Li) 20 Sep 2024
https://hal.science/hal-04703221v1
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[hal-04699429] Phage-mediated intercellular CRISPRi for biocomputation in bacterial consortia
Coordinated actions of cells in microbial communities and multicellular organisms enable them to perform complex tasks otherwise difficult for single cells. This has inspired biological engineers to build cellular consortia for larger circuits with improved functionalities, while implementing communication systems for coordination among cells. Here, we investigate the signalling dynamics of a phage-mediated synthetic DNA messaging system, and couple it with CRISPR interference to build distributed circuits that perform logic gate operations in multicellular bacterial consortia. We find that growth phases of both sender and receiver cells, as well as resource competition between them, shape communication outcomes. Leveraging the easy programmability of DNA messages, we build 8 orthogonal signals and demonstrate that intercellular CRISPRi (i-CRISPRi) regulates gene expression across cells. Finally, we multiplex the i-CRISPRi system to implement several multicellular logic gates that involve up to 7 cells and take up to 3 inputs simultaneously, with single- and dual-rail encoding: NOT, YES, AND, and AND-AND-NOT. The communication system developed here lays the groundwork for implementing complex biological circuits in engineered bacterial communities, using phage signals for communication.
ano.nymous@ccsd.cnrs.fr.invalid (Abhinav Pujar) 16 Sep 2024
https://hal.science/hal-04699429v1
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[hal-04673511] Artificial Intelligence Methods and Models for Retro-Biosynthesis: A Scoping Review
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ano.nymous@ccsd.cnrs.fr.invalid (Guillaume Gricourt) 20 Aug 2024
https://hal.science/hal-04673511v1
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[hal-04644779] Exploring the richness of the French Galaxy Ecosystem
The French Bioinformatics Community has embraced Galaxy since its inception, with a pivotal moment being the Galaxy Tour de France led by Nate Coroar, Anton Nekrutenko, and James Taylor in 2012. This adoption has led to the establishment of over 10 Galaxy servers across France, catering to diverse local needs and specialized thematic areas such as ecology, biodiversity, NGS, proteomics, and more. Among these servers, UseGalaxy.fr stands out as the flagship national instance, launched in 2021 and hosted by the French Institute for Bioinformatics (IFB - ELIXIR-FR). With robust infrastructure boasting 8300 CPU cores, 52 TB of RAM, and GPU cards, UseGalaxy.fr offers a comprehensive suite of over 3,000 tools, including interactive options like Jupyter Notebook, AlphaFold, and Helixer. Notably, it has garnered over 6,000 users who have collectively executed over 3.6 million jobs. Moreover, UseGalaxy.fr hosts specialized subdomains catering to various community needs, such as ecology, metabarcoding, and COVID-19 research, with ongoing integration of new subdomains. The community's commitment to collaboration and consolidation is evident as several local servers have migrated to UseGalaxy.fr in recent years, with others expressing interest in doing the same. The French Galaxy community is deeply engaged in a multitude of projects at national, European, and global levels, including EOSC FAIR EASE, EuroScienceGateway, ATLASea and ABRomics. To foster cohesion and synergy within the community, a Galaxy Working Group led by the French Bioinformatics Institute facilitates regular interactions. This group serves to connect Galaxy users across France, share knowledge, support UseGalaxy.fr, and combat misconceptions about Galaxy within the French scientific community. In this poster presentation, we provide an overview of the dynamic French Galaxy ecosystem, highlighting its diverse servers, engaged researchers, ongoing projects, and collaborative efforts. Through this exploration, we aim to showcase the vibrancy and impact of Galaxy within the French bioinformatics landscape.
ano.nymous@ccsd.cnrs.fr.invalid (Bérénice Batut) 11 Jul 2024
https://hal.science/hal-04644779v1
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[hal-04603192] Artificial Intelligence Methods and Models for Retro-Biosynthesis
Retrosynthesis aims to efficiently plan the synthesis of desirable chemicals by strategically breaking down molecules into readily available building block compounds. Having a long history in chemistry, retro-biosynthesis has also been used in the fields of biocatalysis and synthetic biology. Artificial intelligence (AI) is driving us towards new frontiers in synthesis planning and the exploration of chemical spaces, arriving at an opportune moment for promoting bioproduction that would better align with green chemistry, enhancing environmental practices. In this review, we summarize the recent advancements in the application of AI methods and models for retrosynthetic and retro-biosynthetic pathway design. These techniques can be based either on reaction templates or generative models and require scoring functions and planning strategies to navigate through the retrosynthetic graph of possibilities. We finally discuss limitations and promising research directions in this field.
ano.nymous@ccsd.cnrs.fr.invalid (Guillaume Gricourt) 06 Jun 2024
https://hal.science/hal-04603192v1
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[hal-04592530] Importance of Dynamic Effects in Isobutanol to Linear Butenes Conversion Catalyzed by Acid Zeolites Assessed by AIMD
Dehydration of alcohols into alkenes is a key reaction for the production of fuels and chemicals from biomass. However, the mechanism of these reactions is highly questionable, hindering the rational optimization of efficient catalysts. In the present work, the formation of linear butenes starting from isobutanol catalyzed by proton-exchanged zeolites is unraveled by ab initio molecular dynamics (AIMD). Comparison with static calculations done for a gas phase reaction catalyzed by a proton and for the prototypical chabazite zeolite framework shows that AIMD estimations of the free energy barriers are significantly different from the static ones. Moreover, a common transition state (TS) is found for two competing reactions, namely, the isomerization of isobutanol into butan-2-ol (the dehydration of the latter yielding linear butenes) and the synchronous dehydration and isomerization of isobutanol into products related to linear butenes in a single step. The existence of a post-TS bifurcation prevents a traditional estimation of rates by transition state theory. To circumvent this problem, we quantify relative transmission coefficients using the Bennett–Chandler theory, which shows a clear tendency for decrease of relative frequency for isobutanol isomerization and increase of that for synchronous dehydration and isomerization when switching from 100 to 500 K. This work represents a step forward for the accurate determination of rates for key reactions in alcohol dehydration reactions.
ano.nymous@ccsd.cnrs.fr.invalid (Monika Gešvandtnerová) 29 May 2024
https://ifp.hal.science/hal-04592530v1
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[hal-04504311] Reference‐Quality Free Energy Barriers in Catalysis from Machine Learning Thermodynamic Perturbation Theory
For the first time, we report calculations of the free energies of activation of cracking and isomerization reactions of alkenes that combine several different electronic structure methods with molecular dynamics simulations. We demonstrate that the use of a high level of theory (here Random Phase Approximation—RPA) is necessary to bridge the gap between experimental and computed values. These transformations, catalyzed by zeolites and proceeding via cationic intermediates and transition states, are building blocks of many chemical transformations for valorization of long chain paraffins originating, e.g., from plastic waste, vegetable oils, Fischer–Tropsch waxes or crude oils. Compared with the free energy barriers computed at the PBE+D2 production level of theory via constrained ab initio molecular dynamics, the barriers computed at the RPA level by the application of Machine Learning thermodynamic Perturbation Theory (MLPT) show a significant decrease for isomerization reaction and an increase of a similar magnitude for cracking, yielding an unprecedented agreement with the results obtained by experiments and kinetic modeling.
ano.nymous@ccsd.cnrs.fr.invalid (Jérôme Rey) 14 Mar 2024
https://ifp.hal.science/hal-04504311v1
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[hal-04462025] Selection, adaptation and characterization of electrosynthetic microbial communities
In the context of the energy transition, the use of organic waste in environmental biorefineries is an attractive option due to its low cost and potential to replace fossil fuels. However, this approach presents technological challenges due to the heterogeneity and variability over time of organic waste, increasing the complexity of purification treatments for the molecules of interest and resulting in higher final costs. Microbial electrochemical technologies are emerging as promising solutions to overcome these problems, by allowing the physical separation of oxidation from contaminated waste streams used as raw materials (bioanode) from the synthesis of bio-based chemical molecules (biocathode). Recent scientific studies have focused on the production of methane and acetic acid at the cathode, associated with the electrolysis of water at the anode (abiotic). The objective of this study is to combine a bioanode with a biocathode, in order to reduce carbon dioxide to carboxylates, by the selection of homoacetogenic bacterial communities, at the cathode. The electrons generated at the anode, by oxidation of the organic matter by electrogenic microorganisms, are transferred to the cathode, for the reduction of carbon dioxide into multicarbon molecules by electrotrophic microorganisms. In addition to the two main compartments, an intermediate compartment, isolated by ion exchange membranes, is integrated in order to extract and concentrate the carboxylates produced in a sterile solution. This research aims to explore the diversity of carboxylates produced at the biocathode as a function of the selection and dispersion conditions of the microbial assembly. Both compartments are inoculated with the same mixture of biowaste hydrolysate (Tian et al., 2023) and salt marsh sediments, as hypersaline inocula have been identified as particularly suitable for microbial electrochemical applications. These inoculation conditions make it possible to enrich microbial diversity and study the selection process that takes place at the anode and cathode on the same reservoir of diversity. Only the mixture intended for cathode inoculation is heat-treated in order to select homoacetogenic bacterial communities capable of sporulation, from the class Clostridia (Diallo et al., 2021). Anaerobic conditions are ensured by N2 bubbles and the cathode is supplied daily with CO2, which is the only source of external carbon. In addition, the two electrodes have distinct geometrics: the anode is a carbon cloth while the cathode is a carbon brush with granules of the same material. Thus, the same inoculum is subject to different selection processes, including oxidation or reduction capacity, different carbon sources, the ability to form a biofilm and to carry out electron exchanges with a 2D electrode or 3D, etc. The main objective of this first triplicata experiment is to evaluate the efficiency of the use of a hypersaline inoculum, both for the oxidation of organic matter at the anode, and for the reduction of carbon dioxide to carboxylates at the cathode, as well as the efficiency of the extraction of these carboxylate ions in an intermediate compartment. We also want to determine all the carboxylates produced, in order to evaluate the possibility of diversifying or, on the contrary, of specializing the synthesis of these molecules by modifying the microbial assembly through selection and dispersion. Ultimately, this project aims to guide the production of dyes by a model microorganism, froms carboxylates formed in the biocathode and concentrated within the intermediate compartment. These dyes will have specific applications in the textile industry, demonstrating the potential of this innovative approach in the development of sustainable processes for industrial applications. References: Diallo, M., Kengen, S. W. M., & López-Contreras, A. M. (2021). Sporulation in solventogenic and acetogenic clostridia. Applied Microbiology and Biotechnology, 105(9), 3533-3557. https://doi.org/10.1007/s00253-021-11289-9 Tian, J.-H., Lacroix, R., Yaqoob, A. A., Bureau, C., Midoux, C., Desmond-Le Quéméner, E., & Bouchez, T. (2023). Study of a Pilot Scale Microbial Electrosynthesis Reactor for Organic Waste Biorefinery. Energies, 16(2), 591. https://doi.org/10.3390/en16020591
ano.nymous@ccsd.cnrs.fr.invalid (Louise Rigaud) 16 Feb 2024
https://hal.inrae.fr/hal-04462025v1
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[hal-04351679] Galaxy-SynBioCAD — An Automated Pipeline for Synthetic Biology Design and Engineering
There’s a substantial number of tools released around Synthetic Biology and Metabolic Engineering- related questions and needs. This population of tools is difficult to comprehend and use together, main reasons being complexity and interoperability issues. Indeed, a high level of expertise could be required for installing codes, and execution for real life use cases could be computationally resource demanding. Plus some tools, although complementary, use different inputs and outputs which prevent easy chaining. The SynBioCAD-Galaxy portal [1] is a growing toolshed for synthetic biology, metabolic engineering, and industrial biotechnology. The tools and workflows currently shared on the portal enable one to build libraries of strains producing desired chemical targets covering an end-to-end metabolic pathway design and engineering process: from the selection of strains and targets, the design of DNA parts to be assembled, to the generation of scripts driving liquid handlers for plasmid assembly and strain transformations. Tools are made available on GitHub, anaconda.org and the Galaxy Tool Shed, opening to the greatest number access and utilization throughout the SynBio community, and significant effort has been granted for adopting FAIR principles. As a community effort helped by funded projects, the scope covered by tools is expected to expand over time. The poster will give an overview of the SynBioCAD-Galaxy portal in the context of prediction and construction of E. coli lycopene-producing pathways. The poster will open the discussion around good practices guiding releases of tools through continuous integration. A – lightweight – testing instance of SynBioCAD-Galaxy is available at https://galaxysynbiocad.org.
ano.nymous@ccsd.cnrs.fr.invalid (Joan Hérisson) 18 Dec 2023
https://hal.science/hal-04351679v1
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[hal-04281075] Dryland endolithic Chroococcidiopsis and temperate fresh water Synechocystis have distinct membrane lipid and photosynthesis acclimation strategies upon desiccation and temperature increase
An effect of climate change is the expansion of drylands in temperate regions, predicted to affect microbial biodiversity. Photosynthetic organisms being at the base of ecosystem’s trophic networks, we compared an endolithic desiccation-tolerant Chroococcidiopsis cyanobacteria isolated from gypsum rocks in the Atacama Desert, with a freshwater desiccation-sensitive Synechocystis. We sought whether some acclimation traits in response to desiccation and temperature variations were shared, to evaluate the potential of temperate species to possibly become resilient to future arid conditions. When temperature varies, Synechocystis tunes the acyl composition of its lipids, via a homeoviscuous acclimation mechanism known to adjust membrane fluidity, whereas no such change occurs in Chroococcidiopsis. Vice versa, a combined study of photosynthesis and pigment content shows that Chroococcidiopsis remodels its photosynthesis components and keeps an optimal photosynthetic capacity at all temperatures, whereas Synechocystis is unable to such adjustment. Upon desiccation on a gypsum surface, Synechocystis is rapidly unable to revive, whereas Chroococcidiopsis is capable to recover after three weeks. Using X-ray diffraction, we found no evidence that Chroococcidiopsis could use water extracted from gypsum crystal in such conditions, as a surrogate of missing water. The sulfolipid sulfoquinovosyldiacylglycerol becomes the prominent membrane lipid in both dehydrated cyanobacteria, highlighting an overlooked function for this lipid. Chroococcidiopsis keeps a minimal level of monogalactosyldiacylglycerol, which may be essential for the recovery process. Results support that two independent adaptation strategies have evolved in these species to cope with temperature and desiccation increase, and suggest some possible scenarios for microbial biodiversity change triggered by climate change.
ano.nymous@ccsd.cnrs.fr.invalid (Damien Douchi) 12 Nov 2023
https://hal.science/hal-04281075v1
