Analyse systémique du métabolisme carboné et énergétique de Chlamydomonas reinhardtii

La thèse porte sur le calcul des flux métaboliques de Chlamydomonas reinhardtii, la microalgue modèle, en conditions photoautotrophes dans le but de comprendre la conversion et le stockage de l'énergie. Ce travail a nécessité le développement d'outils méthodologiques incluant (i) la reconstruction d'un réseau métabolique à l'échelle du génome, nommé iAM557, comprenant 557 gènes pour 532 métabolites liés de manière stoechiométrique par 599 réactions métaboliques et (ii) la conception d'un photobioréacteur spécifique à la réalisation d'expériences de marquage au carbone 13 en régime isotopique non stationnaire. Deux approches de calcul des flux métaboliques développées, fondées soit sur une stratégie sous contraintes soit sur l'exploitation de profils d'enrichissements isotopiques collectés au cours des expériences de marquage isotopique, ont permis de modéliser le comportement du système vivant en conditions de croissance pour différentes densités de flux de photons incidentes (200 et 400 mol/m-2s-1) et taux de dilution. Le métabolisme en croissance a pu être décomposé en trois processus responsables de la croissance de la biomasse, de la maintenance cellulaire et d'un mécanisme futile . Plusieurs scénarios métaboliques probables sont avancés pour expliquer cette dissipation d'énergie sans synthèse de biomasse en présence d'une zone sombre dans le volume réactionnel. L'étude de l'effet d'une restriction azotée (carence et limitation) a mis en évidence une remobilisation des réserves macromoléculaires de la microalgue vers la synthèse de composés glucidiques et notamment d'amidon, une molécule d'intérêt bioénergétique.The presented thesis deals with metabolic flux computation of the model microalgae Chlamydomonas reinhardtii under photoautotrophic conditions, in order to understand energy storage and conversion. This work needed the development of methodological tools, such as (i) a genome-scale metabolic network reconstruction, named iAM557, including 557 genes for 532 metabolites connected stoichiometrically by 599 metabolic reactions, and (ii) the design of a specific photobioreactor devoted to isotopic non stationary carbon 13 labeling experiment. Both metabolic flux computation methods, based either on constraints or on isotopic enrichment profiles collected during isotopic labeling experiments, allowed to model living system behavior during growth for different incident photon flux densities (200 and 400 mol/m-2s-1) and dilution rates. Growth metabolism was split up into three processes responsible for biomass growth, cell maintenance and a futile mechanism. Several proposed metabolic scenarios might explain this energy dissipation without biomass synthesis when a dark zone within the reaction volume occured. The study of nitrogen restriction (deprivation and limitation) featured microalgae macromolecular stock restructuration to synthesize carbohydrates compounds and especially starch, a molecule potientially used as bionergy.NANTES-BU Sciences (441092104) / SudocSudocFranceF

How metabolomics can contribute to bio-processes: a proof of concept study for biomarkers discovery in the context of nitrogen-starved microalgae grown in photobioreactors

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Photobioreactor design for isotopic non-stationary 13 C-metabolic flux analysis (INST 13 C-MFA) under photoautotrophic conditions

International audienceAdaptive metabolic behavior of photoautotrophic microorganisms toward genetic and environmental perturbations can be interpreted in a quantitative depiction of carbon flow through a biochemical reaction network using isotopic non-stationary 13C-metabolic flux analysis (INST 13C-MFA). To evaluate 13C-metabolic flux maps for Chlamydomonas reinhardtii, an original experimental framework was designed allowing rapid, reliable collection of high-quality isotopomer data against time. It involved (i) a short-time 13C labeling injection device based on mixing control in a torus-shaped photobioreactor with plug-flow hydrodynamics allowing a sudden step-change in the 13C proportion in the substrate feed and (ii) a rapid sampling procedure using an automatic fast filtration method coupled to a manual rapid liquid nitrogen quenching step. 13C-substrate labeling enrichment was controlled through the total dissolved inorganic carbon concentration in the pulsed solution. First results were obtained from steady-state continuous culture measurements allowing the characterization of the kinetics of label incorporation into light-limited growing cells cultivated in a photobioreactor operating at the maximal biomass productivity for an incident photon flux density of 200 mu molm-2s-1. 13C label incorporation was measured for 21 intracellular metabolites using IC-MS/MS in 58 samples collected across a labeling experiment duration of 7 min. The fastest labeling rate was observed for 2/3-phosphoglycerate with an apparent isotopic stationary state reached after 300s. The labeling rate was consistent with the optimized mixing time of about 4.9s inside the reactor and the shortest reliable sampling period assessed at 5s. Biotechnol. Bioeng. 2012; 109: 30303040. (C) 2012 Wiley Periodicals, Inc