DS10 - Défi des autres savoirs

In vivo determination of enzymatic parameters in a multistep synthetic pathway – ENZINVIVO

Submission summary

Enzyme reactions have long been analyzed in vitro, using pure enzymes and diluted buffer conditions. Due to the large amount of data generated and collected with thousands of enzymes, enzymology has made tremendous progress on understanding the incredible power of biocatalysts. However, dilute, in vitro conditions are far from the surroundings of natural enzymatic reactions that take place inside cells. The cellular medium is more accurately described as a heterogeneous crowded gel, dense and filled with all sorts of macromolecules and cellular lipidic organelles which may result in some partitioning effects and changes in diffusion. Therefore, enzymatic parameters determined using classical enzymology setups may not perfectly represent the real, in vivo based, rate and equilibrium constants. Although some advances have been made toward the comprehension of viscosity and crowding effects, we are still far to derive rate and equilibrium parameters from in vivo enzymatic reactions. The ENZINVIVO project will combine the most up-to-date and advanced techniques in enzymology, synthetic biology, systems biology and theoretical modelling to setup an advanced system capable of mimicking substrate and enzyme variation in classical enzymology.
To exemplify our approach, we will use two isoforms of phytoene synthase (carotenoid biosynthesis) as model enzymes for several reasons. First, carotenoid biosynthesis pathway can be reconstituted in microorganisms such as yeast which will allow the use of genetic engineering tools. Second, phytoene synthase is the pivotal enzyme in the carotenoid pathway, converting the natural soluble precursor geranylgeranyl pyrophosphate (GGPP) into phytoene, the first lipophilic carotenoid molecule. This enzyme is therefore perfectly suited to study a complex enzymatic step for which the cell ultrastructure is of crucial importance. Our project will then consist of a series of increasing complexity experiments, combining simple to complex in vitro experiments, in vivo modulation of substrate and enzyme concentration and analyzing the biological data with mathematical models integrating the critical parameters mentioned in the previous paragraph. In vitro experiments, performed in media of increasing complexity (viscosity, crowding and partial cell structures) will generate pseudo enzymatic constants integrating the complexity factors. Metabolic engineering, by providing advanced techniques to smoothly control gene expression, will allow the construction of a set of yeast strains with: (1) defined (but variables) amounts of GGPP, mimicking the variation of substrate concentration; (2) variable amounts of phytoene synthase, mimicking the variation of enzyme concentration; and (3) absence of presence of the full enzymatic pathway, mimicking the displacement of the thermodynamic equilibrium. In vivo experiments will be performed using the state-of-the-art techniques in systems biology (parallelized chemostats, fast gene regulation, 13C labelling, metabolomics, fluxomics and mathematical/statistical analysis) to determine the in vivo phytoene synthase enzymatic parameters.
Although the results of our project will be derived from a single enzymatic reaction, the developed tools and methods will be quite generic and therefore will serve as a first demonstration that synthetic biology and genetic engineering can trigger new developments in enzymology, challenging the in vitro conceptual field and ultimately proposing new concepts for studying the complexity of enzymatic reactions inside the cell. Furthermore our approach will define the optimal concentrations of substrate and enzymes to achieve for maximum efficiency and perfect homeostasis of the synthetic and natural metabolic pathways. These metabolic engineering tools should then be useful for the scientific community to improve the production, by microorganisms, of natural and synthetic molecules.

Project coordinator

Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (Laboratoire public)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Laboratoire Interdisciplinaire de Physique
Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés
Sciences pour l'Oenologie
Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés

Help of the ANR 651,566 euros
Beginning and duration of the scientific project: December 2016 - 36 Months

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