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

Organisms are either heterotrophic or autotrophic, meaning that they cover their carbon requirements by assimilating organic compounds or by fixing inorganic carbon dioxide (CO). The conversion of a heterotrophic organism into an autotrophic one by metabolic engineering is a long-standing goal in synthetic biology and biotechnology, because it ultimately allows for the production of value-added compounds from CO. The heterotrophic Alphaproteobacterium Methylobacterium extorquens AM1 is a platform organism for a future C1-based bioeconomy. Here we show that M. extorquens AM1 provides unique advantages for establishing synthetic autotrophy, because energy metabolism and biomass formation can be effectively separated from each other in the organism. We designed and realized an engineered strain of M. extorquens AM1 that can use the C1 compound methanol for energy acquisition and forms biomass from CO by implementation of a heterologous Calvin-Benson-Bassham (CBB) cycle. We demonstrate that the heterologous CBB cycle is active, confers a distinct phenotype, and strongly increases viability of the engineered strain. Metabolic C-tracer analysis demonstrates the functional operation of the heterologous CBB cycle in M. extorquens AM1 and comparative proteomics of the engineered strain show that the host cell reacts to the implementation of the CBB cycle in a plastic way. While the heterologous CBB cycle is not able to support full autotrophic growth of M. extorquens AM1, our study represents a further advancement in the design and realization of synthetic autotrophic organisms.

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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.

Based on recent findings indicating that metabolism might be governed by a limit on the rate at which cells can dissipate Gibbs energy, in this Perspective, we propose a new mechanism of how metabolic activity could globally regulate biomolecular processes in a cell. Specifically, we postulate that Gibbs energy released in metabolic reactions is used to perform work, allowing enzymes to self‐propel or to break free from supramolecular structures. This catalysis‐induced enzyme movement will result in increased intracellular motion, which in turn can compromise biomolecular functions. Once the increased intracellular motion has a detrimental effect on regulatory mechanisms, this will establish a feedback mechanism on metabolic activity, and result in the observed thermodynamic limit. While this proposed explanation for the identified upper rate limit on cellular Gibbs energy dissipation rate awaits experimental validation, it offers an intriguing perspective of how metabolic activity can globally affect biomolecular functions and will hopefully spark new research.