Jeudi, 25 avril⋅13:00
Séminaire Denis Jallet & Pierre Millard (Metasys)
Lieu : 401
JALLET Denis Chargé de recherche, INRAE, MetaSys
Titre: The native bacterial microcompartment of Escherichia coli.
Asbtract: Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although 20% of sequenced bacterial genomes contain operons encoding putative BMCs, none have been characterized in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMC were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stochiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome scale modelling and 13C-fluxomics. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli. These results improve our understanding of compartmentalization in a widely used bacterial chassis.
Pierre Millard Chargé de recherche, INRAE, MetaSys
Titre: Combining systems and synthetic biology for in vivo enzymology
Abstract: Enzymatic parameters are classically determined in vitro, under reaction conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We will present a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic pathway aiming to produce phytoene and lycopene in Saccharomyces cerevisiae, we focused on four membrane-associated enzymes, a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We successfully determined in vivo-equivalent Michaelis-Menten parameters (KM, Vmax and kcat) for the phytoene synthase and two of the three phytoene desaturases from quantitative metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Our approach demonstrates the feasibility of assessing enzymatic parameters along a pathway of interest directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.
Lundi, 3 juin⋅14:00
Soutenance HDR Brice Enjalbert
salle 401
Thème général sera la réponse adaptative des microorganismes.
