No abstract

While the stoichiometry of metabolism is probably the best studied cellular level, the dynamics in metabolism can still not be well described, predicted and, thus, engineered. Unknowns in the metabolic flux behavior arise from kinetic interactions, especially allosteric control mechanisms. While the stoichiometry of enzymes is preserved in vitro, their activity and kinetic behavior differs from the in vivo situation. Next to this challenge, it is infeasible to test the interaction of each enzyme with each intracellular metabolite in vitro exhaustively. As a consequence, the whole interacting metabolome has to be studied in vivo to identify the relevant enzymes properties. In this review we discuss current approaches for in vivo perturbation experiments, that is, stimulus response experiments using different setups and quantitative analytical approaches, including dynamic carbon tracing. Next to reliable and informative data, advanced modeling approaches and computational tools are required to identify kinetic mechanisms and their parameters.

Background: Microbial metabolism is highly dependent on the environmental conditions. Especially, the substrate concentration, as well as oxygen availability, determine the metabolic rates. In large-scale bioreactors, microorganisms encounter dynamic conditions in substrate and oxygen availability (mixing limitations), which influence their metabolism and subsequently their physiology. Earlier, single substrate pulse experiments were not able to explain the observed physiological changes generated under large-scale industrial fermentation conditions. Results: In this study we applied a repetitive feast-famine regime in an aerobic Escherichia coli culture in a timescale of seconds. The regime was applied for several generations, allowing cells to adapt to the (repetitive) dynamic environment. The observed response was highly reproducible over the cycles, indicating that cells were indeed fully adapted to the regime. We observed an increase of the specific substrate and oxygen consumption (average) rates during the feast-famine regime, compared to a steady-state (chemostat) reference environment. The increased rates at same (average) growth rate led to a reduced biomass yield (30% lower). Interestingly, this drop was not followed by increased by-product formation, pointing to the existence of energy-spilling reactions. During the feast-famine cycle, the cells rapidly increased their uptake rate. Within 10 s after the beginning of the feeding, the substrate uptake rate was higher (4.68 μmol/g CDW /s) than reported during batch growth (3.3 μmol/g CDW /s). The high uptake led to an accumulation of several intracellular metabolites, during the feast phase, accounting for up to 34% of the carbon supplied. Although the metabolite concentrations changed rapidly, the cellular energy charge remained unaffected, suggesting well-controlled balance between ATP producing and ATP consuming reactions. Conclusions: The adaptation of the physiology and metabolism of E. coli under substrate dynamics, representative for large-scale fermenters, revealed the existence of several cellular mechanisms coping with stress. Changes in the substrate uptake system, storage potential and energy-spilling processes resulted to be of great importance. These metabolic strategies consist a meaningful step to further tackle reduced microbial performance, observed under large-scale cultivations.

7Background 8 Microbial metabolism is highly dependent on the environmental conditions. Especially, the 9 substrate concentration, as well as oxygen availability, determine the metabolic rates. In large-10 scale bioreactors, microorganisms encounter dynamic conditions in substrate and oxygen 11 availability (mixing limitations), which influence their metabolism and subsequently their 12 physiology. Earlier, single substrate pulse experiments were not able to explain the observed 13 physiological changes generated under large-scale industrial fermentation conditions. 14 Results 15In this study we applied a repetitive feast-famine regime in an aerobic Escherichia coli culture 16 in a time-scale of seconds. The regime was applied for several generations, allowing cells to 17 adapt to the (repetitive) dynamic environment. The observed response was highly reproducible 18 over the cycles, indicating that cells were indeed fully adapted to the regime. We observed an 19 increase of the specific substrate and oxygen consumption (average) rates during the feast-20 famine regime, compared to a steady-state (chemostat) reference environment. The increased 21 rates at same (average) growth rate led to a reduced biomass yield (30% lower). Interestingly, 22 this drop was not followed by increased by-product formation, pointing to the existence of 23 energy-spilling reactions and/or less effective ATP synthesis. During the feast-famine cycle, 24 the cells rapidly increased their uptake rate. Within 10 seconds after the beginning of the 25 feeding, the substrate uptake rate was higher (4.68 μmol/gCDW/s) than reported during batch 26 growth (3.3 μmol/gCDW/s). The high uptake led to an accumulation of several intracellular 27 metabolites, during the feast phase, accounting for up to 34 % of the carbon supplied. Although 28 the metabolite concentrations changed rapidly, the cellular energy charge remained unaffected, 29 3 suggesting well-controlled balance between ATP producing and ATP consuming reactions. The 30 role of inorganic polyphosphate as an energy buffer is discussed. 31 Conclusions 32The adaptation of the physiology and metabolism of Escherichia coli under substrate dynamics, 33 representative for large-scale fermenters, revealed the existence of several cellular mechanisms 34 coping with stress. Changes in the substrate uptake system, storage potential and energy-spilling 35 processes resulted to be of great importance. These metabolic strategies consist a meaningful 36 step to further tackle reduced microbial performance, observed under large-scale cultivations. 37 38 Keywords 39 Escherichia coli; feast-famine; substrate dynamics; dynamic metabolic responses; energy 40 homeostasis 41 4 Introduction 42Microorganisms are widely used for the production of chemicals, ranging from small organic 43 acids to large proteins, including biopharmaceuticals, biochemicals and bulk biofuels [1][2][3]. In 44 order to meet the cost targets and demands, large-scale production cultivations are and will be 45 required [4]. ...