Contrôle spatio-temporel de la coopération chez des communautés de levures

Les communautés microbiennes naturelles sont souvent structurées spatialement, avec différentes espèces qui interagissent et se développent de manière hétérogène. Même dans le cas des colonies clonales, le microenvironnement individuel des cellules est affecté par la consommation des nutriments, l'excrétion de molécules inhibitrices ou les communications chimiques, menant à une différentiation phénotypique entre les cellules. Dans les domaines de l'écologie et de l'évolution microbienne, l'organisation spatiale joue un rôle déterminant dans le destin d'une population. Malgré les récents travaux portant sur le design rationnel de communautés microbiennes, il y a par contre peu de méthodologies établies pour contrôler, surtout dans l'espace, ces associations microbiennes. L'objectif de ma thèse est de mieux comprendre le rôle de l'organisation spatiale dans les interactions des communautés microbiennes. Pour se faire j'ai construit et optimisé une souche de levure Saccharomyces cerevisiae pour le control optogénétique de la production d'invertase SUC2, permettant d'induire la production locale de bien commun avec de la lumière bleue. J'ai construit un équipement expérimental sur mesure pour créer avec la lumière et observer au fil du temps des patterns de cellules coopératrices/tricheuses et je l'ai utilisé pour étudier l'influence de l'organisation spatiale sur le développement de ce genre de consortia. J'ai trouvé que ce consortium de coopérateur/tricheur se comporte comme un filtre spatial passe-bande, filtrant les fluctuations spatiales courtes de phénotypes coopérateur/tricheur mais aussi les larges régions de phénotypes identiques. Les résultats présentés dans ce travail montrent l'importance de la structuration spatiotemporelle dans les systèmes coopératifs, due aux gradients de molécules diffusibles et à l'hétérogénéité phénotypique dans la population de cellules. Cela représente un pas vers une meilleure caractérisation des échelles spatiales des interactions microbiennes qui peut servir à approfondir notre compréhension des microbiomes naturels et à aider le design de microbiomes synthétiques.Natural microbial communities can often be spatially structured, with different species that interact and grow in a heterogeneous manner. Even among clonal colonies, nutrient uptake, inhibitory chemicals excretion or chemical communication considerably affect the individual cell microenvironment leading to cell-to-cell phenotypic differentiation. In both microbial ecology and evolution, spatial organization plays a key role in the population fate. While recent works are progressing in the rational design of microbial communities, there are few established methodologies to control, especially spatially, the functioning and development of microbial ecosystems. My PhD aims to get new insights in the role of spatial organisation in the interactions of microbial communities. To this end I designed and optimised a Saccharomyces cerevisiae yeast strain for optogenetic control of the SUC2 invertase production, allowing to induce the local production of public goods with blue light. I built a custom experimental device to create with light and observe through time patterns of cooperator/cheater cells and used it to investigate the influence of spatial organisation on the development of such consortia. I found that this cooperator/cheater consortium acts as a spatial bandpass filter, filtering out short spatial fluctuations of cheater/cooperator phenotypes but also large areas of identical phenotypes. The results presented in this work show the importance of the spatiotemporal structuration in a cooperative system, due to gradients of diffusive molecules and phenotypic heterogeneity in the cell population. This represents a step toward better characterisations of microbial interaction length scales which can serve to deepen our understanding of natural microbiomes and to help designing synthetic ones

Contrôle spatio-temporel de la coopération chez des communautés de levures

Natural microbial communities can often be spatially structured, with different species that interact and grow in a heterogeneous manner. Even among clonal colonies, nutrient uptake, inhibitory chemicals excretion or chemical communication considerably affect the individual cell microenvironment leading to cell-to-cell phenotypic differentiation. In both microbial ecology and evolution, spatial organization plays a key role in the population fate. While recent works are progressing in the rational design of microbial communities, there are few established methodologies to control, especially spatially, the functioning and development of microbial ecosystems. My PhD aims to get new insights in the role of spatial organisation in the interactions of microbial communities. To this end I designed and optimised a Saccharomyces cerevisiae yeast strain for optogenetic control of the SUC2 invertase production, allowing to induce the local production of public goods with blue light. I built a custom experimental device to create with light and observe through time patterns of cooperator/cheater cells and used it to investigate the influence of spatial organisation on the development of such consortia. I found that this cooperator/cheater consortium acts as a spatial bandpass filter, filtering out short spatial fluctuations of cheater/cooperator phenotypes but also large areas of identical phenotypes. The results presented in this work show the importance of the spatiotemporal structuration in a cooperative system, due to gradients of diffusive molecules and phenotypic heterogeneity in the cell population. This represents a step toward better characterisations of microbial interaction length scales which can serve to deepen our understanding of natural microbiomes and to help designing synthetic ones.Les communautés microbiennes naturelles sont souvent structurées spatialement, avec différentes espèces qui interagissent et se développent de manière hétérogène. Même dans le cas des colonies clonales, le microenvironnement individuel des cellules est affecté par la consommation des nutriments, l'excrétion de molécules inhibitrices ou les communications chimiques, menant à une différentiation phénotypique entre les cellules. Dans les domaines de l'écologie et de l'évolution microbienne, l'organisation spatiale joue un rôle déterminant dans le destin d'une population. Malgré les récents travaux portant sur le design rationnel de communautés microbiennes, il y a par contre peu de méthodologies établies pour contrôler, surtout dans l'espace, ces associations microbiennes. L'objectif de ma thèse est de mieux comprendre le rôle de l'organisation spatiale dans les interactions des communautés microbiennes. Pour se faire j'ai construit et optimisé une souche de levure Saccharomyces cerevisiae pour le control optogénétique de la production d'invertase SUC2, permettant d'induire la production locale de bien commun avec de la lumière bleue. J'ai construit un équipement expérimental sur mesure pour créer avec la lumière et observer au fil du temps des patterns de cellules coopératrices/tricheuses et je l'ai utilisé pour étudier l'influence de l'organisation spatiale sur le développement de ce genre de consortia. J'ai trouvé que ce consortium de coopérateur/tricheur se comporte comme un filtre spatial passe-bande, filtrant les fluctuations spatiales courtes de phénotypes coopérateur/tricheur mais aussi les larges régions de phénotypes identiques. Les résultats présentés dans ce travail montrent l'importance de la structuration spatiotemporelle dans les systèmes coopératifs, due aux gradients de molécules diffusibles et à l'hétérogénéité phénotypique dans la population de cellules. Cela représente un pas vers une meilleure caractérisation des échelles spatiales des interactions microbiennes qui peut servir à approfondir notre compréhension des microbiomes naturels et à aider le design de microbiomes synthétiques

Contrôle spatio-temporel de la coopération chez des communautés de levures

Natural microbial communities can often be spatially structured, with different species that interact and grow in a heterogeneous manner. Even among clonal colonies, nutrient uptake, inhibitory chemicals excretion or chemical communication considerably affect the individual cell microenvironment leading to cell-to-cell phenotypic differentiation. In both microbial ecology and evolution, spatial organization plays a key role in the population fate. While recent works are progressing in the rational design of microbial communities, there are few established methodologies to control, especially spatially, the functioning and development of microbial ecosystems. My PhD aims to get new insights in the role of spatial organisation in the interactions of microbial communities.To this end I designed and optimised a Saccharomyces cerevisiae yeast strain for optogenetic control of the SUC2 invertase production, allowing to induce the local production of public goods with blue light. I built a custom experimental device to create with light and observe through time patterns of cooperator/cheater cells and used it to investigate the influence of spatial organisation on the development of such consortia. I found that this cooperator/cheater consortium acts as a spatial bandpass filter, filtering out short spatial fluctuations of cheater/cooperator phenotypes but also large areas of identical phenotypes. The results presented in this work show the importance of the spatiotemporal structuration in a cooperative system, due to gradients of diffusive molecules and phenotypic heterogeneity in the cell population. This represents a step toward better characterisations of microbial interaction length scales which can serve to deepen our understanding of natural microbiomes and to help designing synthetic ones.Les communautés microbiennes naturelles sont souvent structurées spatialement, avec différentes espèces qui interagissent et se développent de manière hétérogène. Même dans le cas des colonies clonales, le microenvironnement individuel des cellules est affecté par la consommation des nutriments, l’excrétion de molécules inhibitrices ou les communications chimiques, menant à une différentiation phénotypique entre les cellules. Dans les domaines de l’écologie et de l’évolution microbienne, l’organisation spatiale joue un rôle déterminant dans le destin d’une population. Malgré les récents travaux portant sur le design rationnelde communautés microbiennes, il y a par contre peu de méthodologies établies pour contrôler, surtout dans l’espace, ces associations microbiennes.L’objectif de ma thèse est de mieux comprendre le rôle de l’organisation spatiale dans les interactions des communautés microbiennes.Pour se faire j’ai construit et optimisé une souche de levure Saccharomyces cerevisiae pour le control optogénétique de la production d’invertase SUC2, permettant d’induire la production locale de bien commun avec de la lumière bleue. J’ai construit un équipement expérimental sur mesure pour créer avec la lumière et observer au fil du temps des patterns de cellules coopératrices/tricheuses et je l’ai utilisé pour étudier l’influence de l’organisation spatiale sur le développement de ce genre de consortia. J’ai trouvé que ce consortium decoopérateur/tricheur se comporte comme un filtre spatial passe-bande, filtrant les fluctuations spatiales courtes de phénotypes coopérateur/tricheur mais aussi les larges régions de phénotypes identiques. Les résultats présentés dans ce travail montrent l’importance de la structuration spatiotemporelle dans les systèmes coopératifs, due aux gradients de molécules diffusibles et à l’hétérogénéité phénotypique dans la population de cellules. Cela représente un pas vers une meilleure caractérisation des échelles spatiales des interactions microbiennes qui peut servir à approfondir notre compréhension des microbiomes naturels et à aider le design de microbiomes synthétiques

Autonomous and Assisted Control for Synthetic Microbiology

The control of microbes and microbial consortia to achieve specific functions requires synthetic circuits that can reliably cope with internal and external perturbations. Circuits that naturally evolved to regulate biological functions are frequently robust to alterations in their parameters. As the complexity of synthetic circuits increases, synthetic biologists need to implement such robust control “by design”. This is especially true for intercellular signaling circuits for synthetic consortia, where robustness is highly desirable, but its mechanisms remain unclear. Cybergenetics, the interface between synthetic biology and control theory, offers two approaches to this challenge: external (computer-aided) and internal (autonomous) control. Here, we review natural and synthetic microbial systems with robustness, and outline experimental approaches to implement such robust control in microbial consortia through population-level cybergenetics. We propose that harnessing natural intercellular circuit topologies with robust evolved functions can help to achieve similar robust control in synthetic intercellular circuits. A “hybrid biology” approach, where robust synthetic microbes interact with natural consortia and—additionally—with external computers, could become a useful tool for health and environmental applications

The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments

International audienceProgress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories

CyberSco.Py an open-source software for event-based, conditional microscopy

International audienceAbstract Timelapse fluorescence microscopy imaging is routinely used in quantitative cell biology. However, microscopes could become much more powerful investigation systems if they were endowed with simple unsupervised decision-making algorithms to transform them into fully responsive and automated measurement devices. Here, we report CyberSco.Py, Python software for advanced automated timelapse experiments. We provide proof-of-principle of a user-friendly framework that increases the tunability and flexibility when setting up and running fluorescence timelapse microscopy experiments. Importantly, CyberSco.Py combines real-time image analysis with automation capability, which allows users to create conditional, event-based experiments in which the imaging acquisition parameters and the status of various devices can be changed automatically based on the image analysis. We exemplify the relevance of CyberSco.Py to cell biology using several use case experiments with budding yeast. We anticipate that CyberSco.Py could be used to address the growing need for smart microscopy systems to implement more informative quantitative cell biology experiments

Yeast cell responses and survival during periodic osmotic stress are controlled by glucose availability

Natural environments of living organisms are often dynamic and multifactorial, with multiple parameters fluctuating over time. To better understand how cells respond to dynamically interacting factors, we quantified the effects of dual fluctuations of osmotic stress and glucose deprivation on yeast cells using microfluidics and time-lapse microscopy. Strikingly, we observed that cell proliferation, survival, and signaling depend on the phasing of the two periodic stresses. Cells divided faster, survived longer, and showed decreased transcriptional response when fluctuations of hyperosmotic stress and glucose deprivation occurred in phase than when the two stresses occurred alternatively. Therefore, glucose availability regulates yeast responses to dynamic osmotic stress, showcasing the key role of metabolic fluctuations in cellular responses to dynamic stress. We also found that mutants with impaired osmotic stress response were better adapted to alternating stresses than wild-type cells, showing that genetic mechanisms of adaptation to a persistent stress factor can be detrimental under dynamically interacting conditions

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales

ABSTRACT Optogenetics arises as a valuable tool to precisely control genetic circuits in microbial cell factories. Light control holds the promise of optimizing bioproduction methods and maximize yields, but its implementation at different steps of the strain development process and at different culture scales remains challenging. In this study, we aim to control beta-carotene bioproduction using optogenetics in Saccharomyces cerevisiae and investigate how its performance translates across culture scales. We built four lab-scale illumination devices, each handling different culture volumes, and each having specific illumination characteristics and cultivating conditions. We evaluated optogenetic activation and beta-carotene production across devices and optimized them both independently. Then, we combined optogenetic induction and beta-carotene production to make a light-inducible beta-carotene producer strain. This was achieved by placing the transcription of the bifunctional lycopene cyclase / phytoene synthase CrtYB under the control of the pC120 optogenetic promoter regulated by the EL222-VP16 light-activated transcription factor, while other carotenogenic enzymes (CrtI, CrtE, tHMG) were expressed constitutively. We show that illumination, culture volume and shaking impact differently optogenetic activation and beta-carotene production across devices. This enabled us to determine the best culture conditions to maximize light-induced beta-carotene production in each of the devices, reaching a content of up to 880 μg/gCDW. Our study exemplifies the stakes of scaling up optogenetics in devices of different lab scales and sheds light on the interplays and potential conflicts between optogenetic control and metabolic pathway efficiency. As a general principle, we propose that it is important to first optimize both components of the system independently, before combining them into optogenetic producing strains to avoid extensive troubleshooting. We anticipate that our results can help designing both strains and devices that could eventually lead to larger scale systems in an effort to bring optogenetics to the industrial scale

Adeno-Associated Virus Vector (AAV) microdystrophin gene therapy prolongs survival and restores muscle function in the canine model of Duchenne Muscular Dystrophy (DMD)

Duchenne Muscular Dystrophy (DMD) is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno-Associated Virus vectors (rAAV) hold great promise and allow very efficient transduction of skeletal and cardiac muscles. However, full-length dystrophin cDNA exceeds the packaging capacity for a single rAAV gene-delivery cassette. Therefore, truncated versions namely micro-dystrophins have been designed and optimized to contain few clinically important regions of the dystrophin protein. We have tested a rAAV2/8 vector encoding a sequence optimised canine micro-dystrophin transgene, driven by a muscle-synthetic Spc512 promoter (rAAV2/8-Spc512-µDys) in a total of 12 Golden Retriever Muscular Dystrophy (GRMD) dogs, the canine model of DMD. Isolated limb perfusion studies using a single administration of vector induced high levels of micro-dystrophin expression in the treated limb (up to 90% dystrophin positive fibres) with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. Similarly, single-dose intravascular delivery of the same rAAV2/8-Spc512-µDys, in absence of immunosuppression, led to long-term transduction of distant muscle groups and extended lifespan (up to 2 years). Profound improvement of multiple clinical features was observed, including gait and respiratory parameters and no toxicity or deleterious humoral and/or cell-mediated immune responses were observed. This study demonstrates the safety and long term efficacy of rAAV2/8-Spc5.12-µDys gene therapy in a relevant large-animal models of DMD and paves the way towards human clinical gene therapy using systemic peripheral vein administration of vector, and applicable to all DMD patients regardless of their genotype