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

The system we have chosen is a synthetic pathway reconstituted in yeast: the production of carotenoids. We chose to study the enzymatic parameters of the first enzyme of this pathway: phytoene synthase (CrtB), which converts geranyl-geranyl pyrophosphate (GGPP) to phytoene. GGPP, the precursor of the pathway, is produced by the enzyme CrtE. The variation of the amount of GGPP was addressed with synthetic biology tools: promoters of different strengths and integration of multiple copies of the CRTE gene. We constructed about 30 strains producing variable levels of GGPP and constant levels of the CrtB enzyme. We then developed systems biology techniques to analyze precursor and phytoene production. We also analyzed the physiological consequences (transcriptome, targeted proteome) of engineering these strains. Finally, we have built tools to analyze metabolic fluxes and convert these data into enzymatic parameters. The technologies developed in the project (except analytical) are not specific and can be used for other study models.

The project has shown that in vivo determination of the kinetic parameters of an enzyme is possible. This innovative approach is free from the constraints of classical in vitro enzymology. In the context of the production of molecules by microorganisms (metabolic engineering), the tools and concepts developed here will make it possible to adjust the quantities of enzymes participating in the various stages of the synthetic pathway, to avoid the overproduction of potentially toxic intermediates and thus to improve the yield and productivity while maintaining a good cellular homeostasis

Not all of the original objectives have been achieved as of the date of this paper, but the work in progress shows with certainty that they have now been achieved. The essential answer is that it is possible to calculate enzyme constants in a cellular context. The tools put in place assume a certain number of prerequisites that we have described in our work. The use of synthetic biology and systems biology tools in a microbial host allows us to meet the initial specifications: to analyze enzymatic reactions in a cellular context. Even if this context is not exactly that of the initial cell, it still takes into account the effects of the cell structure, the presence of high concentrations of macromolecules and all the factors that can influence the enzymatic activities. To our knowledge, this is the first demonstration that it is possible to calculate these parameters with a modulation of the amount of substrate without addition by the experimenter, thus only limited by what the cell can produce.
The development time of the tools for the project has been long and our results have been impacted by a number of difficulties that we have overcome in recent months. This investment will pay off as we can now report on the catalytic efficiency of the different enzymes in the entire ß-carotene production pathway, so that production is maximized without impacting cell physiology. Furthermore, it is also possible to account for these parameters by changing the localization of the enzymes, which will allow us to understand the effects of cellular compartmentalization. The concepts developed during the project are for us the first brick of a detailed analysis of the enzymatic efficiency of a metabolic pathway.

A first paper on the methodology of metabolite analysis has been published as well as two others dealing with algorithmic developments. Two software packages have been created and are available on the GitHub platform. However, the project is not finished and the last results are being finalized. At least two papers are planned: one on the analysis of the consequences of large variations in GGPP and phytoene production at the transcriptome level and a second on the first description of the obtaining of enzymatic parameters directly in vivo.

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.