biostudies-literatureCastano-Cerezo SAgence Nationale de la Recherche (ANR)Agence Nationale de la Recherche5169-5185https://www.ebi.ac.uk/biostudies/studies/S-EPMC11535393biostudies-literatureUnknown43(21)Enzymatic parameters are classically determined in 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 parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in 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 determined in vivo Michaelis-Menten parameters (K_M, V_max, and k<sub>The EMBO journalCombining systems and synthetic biology for in vivo enzymology.PMC11535393ANR-16-CE11-0022Tholey AHeux SGarcia-Alles LFBellvert FKulyk HTruan GGaleote VCamarasa CTreitz CCastano-Cerezo SChamas AMillard PfalseCombining systems and synthetic biology for in vivo enzymology.Enzymatic parameters are classically determined in 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 parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in 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 determined in vivo Michaelis-Menten parameters (K_M, V_max, and k<sub>2024-01-01T00:00:00Z2024 Nov2026-06-01T20:32:20.416Z2025-04-04T11:43:22.654ZS-EPMC115353933932275710.1038/s44318-024-00251-w