Combining systems and synthetic biology for in vivo enzymology

Abstract

AbstractEnzymatic parameters are classically determinedin vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parametersin vivo. In the context of a synthetic carotenoid pathway inSaccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to studyin vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determinedin vivoMichaelis-Menten parameters (KM,Vmaxandkcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences betweenin vivoandin vitroparameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directlyin vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.