Organization of intracellular reactions with rationally designed scaffolding systems

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THESE 2009

 Title of the project 

Organization of intracellular reactions with rationally designed scaffolding systems

 PhD student

Camille Delebecque

 University-Doctoral school

Frontières du Vivant (FdV), Faculté de Médecine, Paris-Descartes

 Supervisors

-Pamela Silver, System Biology Department, Harvard Medical School, 200 Longwood avenue, Boston MA 02115, USA
- Ariel Lindner, Evolutionary Systems Biology, Faculté de Médecine Paris-Descartes, 24 rue du faubourg Saint-Jacques, 75014 Paris, France

 Laboratories of welcome

- System Biology Department, Harvard Medical School, Boston
- Centre de Recherches Interdisciplinaires, (CRI), Faculté de Médecine , Université Paris-Descartes

 Responsibles for thesis

- Pamela Silver, Boston
- Ariel Lindner, Paris

 Duration

Trois ans (fin 2009 à fin 2012). Thèse soutenue le 15 Novembre 2012 à Paris

Summary

Hydrogen has been considered a potential fuel for the future since it is carbon-free and oxidized to water.
In this project we intend to address the difficulty of biologically producing hydrogen using bacteria as micro and fully automatized factories. Using tools of molecular biology, synthetic biology and genetic engineering we aim at creating within the bacterium cell a localized anaerobic microenvironment suitable for the work of highly oxygen sensitive hydrogenases.
Alongside with the innovative synthetic biology approach, this system has lots of promises concerning hydrogen yields and as well as economically viable industrial applications.

Results

In cells bio-enzymatic pathways are often spatially organized into complexes, into organelles or onto protein scaffolds. Spatial organization limits diffusion and helps channels substrates between enzymatic cores, limiting competing reactions, insulating and increasing yields of sequential metabolic reactions. In this PhD thesis work, we engineered new tools to control the precise spatial organization of enzymes and increase the titer of specific pathways.
We design and engineer “artificial organelles” made of assembling RNA nanostructures. These scaffolds are made out of assembling non-coding RNA molecules we specifically design to polymerize into multi-dimensional nanostructures inside bacterial cells. These structures have docking sites to target enzymes onto them and control their respective distance and stochiometry.
We demonstrate the validity of our approach by optimizing and improving the production of biohydrogen and designing a protocol to simplify and standardize the use of RNA scaffold. Moreover, we develop a new synthetic biology “chassis” by developing strategies to engineer Anabaena PCC7120 and control the spatial localization of metabolic pathway at the cellular level. By targeting specific enzymes into oxygen-depleting heterocysts, metabolic engineers can now implement oxygen-sensitive pathways into oxygen evolving cyanobacteria. This PhD work opens the door to an array of new applications spanning synthetic biology, structural biology to nanotechnology.

Livrables

Publications

  • Camille J Delebecque et al. (2011) Organization of Intracellular Reactions with Rationally Designed RNA Assemblies. Science, 333, 470

  • Camille J Delebecque, Pamela A Silver and Ariel B Lindner (2012) Designing and using RNA scaffolds to assemble proteins in vivo. Nature Protocols, 7, 10, 1797-1807

Contacts

Pamela A. Silver

Harvard Medical School, Boston

pamela_silver@hms.harvard.edu


Ariel Lindner

Faculté de Médecine Paris-Descartes

ariel.lindner@inserm.fr