phone +31 50 363 8146) $ These authors contributed equally to this work The principles governing cellular metabolic operation are poorly understood. Because diverse 3 organisms show similar metabolic flux patterns, we hypothesized that a fundamental 4 thermodynamic constraint might shape cellular metabolism. Here, we developed a constraint-based 5 model for Saccharomyces cerevisiae with a comprehensive description of biochemical 6 thermodynamics including a Gibbs energy balance. Nonlinear regression analyses of quantitative 7 metabolome and physiology data revealed the existence of an upper rate limit for cellular Gibbs 8 energy dissipation. Applying this limit in flux balance analyses with growth maximization as the 9 objective, our model correctly predicted the physiology and intracellular metabolic fluxes for10 different glucose uptake rates as well as the maximal growth rate. We found that cells arrange their 11 intracellular metabolic fluxes in such a way that, with increasing glucose uptake rates, they can 12 accomplish optimal growth rates, but stay below the critical rate limit in Gibbs energy dissipation. 13Once all possibilities for intracellular flux redistribution are exhausted, cells reach their maximal 14 growth rate. This principle also holds for Escherichia coli and different carbon sources. Our work 15 proposes that metabolic reaction stoichiometry, a limit in the cellular Gibbs energy dissipation rate, 16 and the objective of growth maximization shape metabolism across organisms and conditions. 17A key question in metabolic research is to understand how and why cells organize their metabolism, i.e. 18 their fluxes through the metabolic network, in a particular manner. Such understanding is highly relevant 19 from a fundamental point of view, but also should enable us to devise computational methods for 20 metabolic-flux prediction; an important capability for fundamental biology and biotechnology. 21

Metabolome, proteome and physiology measurements were combined with mathematical modeling to unravel the temporal regulation of the metabolic fluxes during the diauxic shift in Saccharomyces cerevisiae.

Eukaryotic cell division is known to be controlled by the cyclin/cyclin dependent kinase (CDK) machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic triggers for cell-cycle initiation and progression. Using microfluidics, cell-cycle reporters, and single-cell metabolite measurements, we found that metabolism of budding yeast is a CDK-independent oscillator that oscillates across different growth conditions, both in synchrony with and also in the absence of the cell cycle. Using environmental perturbations and dynamic single-protein depletion experiments, we found that the metabolic oscillator and the cell cycle form a system of coupled oscillators, with the metabolic oscillator separately gating and maintaining synchrony with the early and late cell cycle. Establishing metabolism as a dynamic component within the cell-cycle network opens new avenues for cell-cycle research and therapeutic interventions for proliferative disorders.

The yeast Saccharomyces cerevisiae can show different metabolic phenotypes (e.g. fermentation and respiration). Based on data from the literature, we argue that the substrate uptake rate is the core variable in the system that controls the global metabolic phenotype. Consequently the metabolic phenotype that the cell expresses is not dependent on the type of the sugar or its concentration, but only on the rate at which the sugar enters the cell. As this requires the cells to 'measure' metabolic flux, we discuss the existing clues toward a flux-sensing mechanism in this organism and also outline several aspects of the involved flux-dependent regulation system. It becomes clear that the sensing and regulation system that divides the taken up carbon flux into the respiratory or fermentative pathways is complex with many molecular components interacting on multiple levels. To obtain a true understanding about how the global metabolic phenotype of S. cerevisiae is controlled by the glucose uptake rate, different tools and approaches from systems biology will be required.

A comprehensive description of the phenotypic changes during cellular aging is key towards unraveling its causal forces. Previously, we mapped age-related changes in the proteome and transcriptome (Janssens et al., 2015). Here, employing the same experimental procedure and model-based inference, we generate a comprehensive account of metabolic changes during the replicative life of Saccharomyces cerevisiae. With age, we found decreasing metabolite levels, decreasing growth and substrate uptake rates accompanied by a switch from aerobic fermentation to respiration, with glycerol and acetate production. The identified metabolic fluxes revealed an increase in redox cofactor turnover, likely to combat increased production of reactive oxygen species. The metabolic changes are possibly a result of the age-associated decrease in surface area per cell volume. With metabolism being an important factor of the cellular phenotype, this work complements our recent mapping of the transcriptomic and proteomic changes towards a holistic description of the cellular phenotype during aging.