Combining systems and synthetic biology for in vivo enzymology

Abstract

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_cat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. 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 directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

Keywords: Biotechnology; Carotenoid Synthesis; Enzymatic Parameters; In Vivo vs In Vitro; Metabolism.

Figure 1

General principle of the proposed strategy for in vivo enzymology and comparison between in vitro and in vivo enzymology approaches.

Figure 2. Scheme of the construction of yeast strains with different GGPP concentrations.

Red filled dots represent a copy of GGPP synthase, and empty dots represent an empty integration cassette.

Figure 3. Construction of the set of yeast strains with different intracellular GGPP concentrations.

(A) Genomic modification scheme for the yENZ15 strain and intracellular fates of GGPP in S. cerevisiae. Overexpressed enzymes are shown in blue, enzyme deletion is shown in red, and enzyme modulation is shown in green. (B) GGPP concentration (green bars) and specific growth rate of S. cerevisiae strains (orange dots). Mean values ± standard deviations (error bars) were estimated from three independent biological replicates. Source data are available online for this figure.

Figure 4. In vivo characterization of phytoene synthase from Pantoea ananas.

(A) Scheme for the biosynthesis of phytoene by CrtB. (B) Steady-state flux and substrate concentration simulated for three different CrtB activities (low, medium and high) under a broad range of GGPP-producing flux. (C) In vivo characterization of CrtB in S. cerevisiae: phytoene production as a function of GGPP concentration in strains with low (TEF1mut2p), medium (PGIp) and high (PDC1p) level of CrtB. Each data point represents an independent biological replicate. For PGIp strains, red line represents the best fit of a Michaelis-Menten rate law, and shaded area corresponds to 95% confidence interval on the fit. Source data are available online for this figure.

Figure 5. In vivo characterization of three phytoene desaturases.

(A) Scheme for the biosynthesis of lycopene by CrtI. (B–D) In vivo characterization of Crtl from P. ananas (B), X. dendrorus (C), and B. trispora (D). Each data point represents an independent biological replicate, red lines represent the best fits of a Michaelis-Menten rate law, and shaded areas correspond to 95% confidence intervals on the fits. Source data are available online for this figure.

Figure EV1. Expression of PaCrtB-FLAG in the different strains.

PaCrtB-FLAG expression determined by western blot in yeast strains with different GGPP concentrations (panels (A–C) correspond to three different biological replicates).

Figure EV2. Lycopene production in S. cerevisiae.

(A) S. cerevisiae yGGPP34 strain expressing PDC1p-PaCrtB and TDH3p-BtCrtI. Bright field images were acquired using the camera LEICA DFC300FX mounted in the microscope Leica DM4000B with Leica EL6000 light source. Lycopene crystals are observed in red. (B) Decrease of specific growth rate in yeast strains expressing BtCrtI with different phytoene concentrations.

Figure EV3. Expression of CrtI in the different strains.

Expression of the three studied CrtI-V5 proteins in strains with low (yGGPP030) and high (yGGPP044) phytoene content (panels (A and B) correspond to three different biological replicates).

Figure EV4. Correlations between OD and biomass concentration.

Correlation between cell/mL and OD_600nm (A) and between OD_600nm and mg DCW (B).