The Role of Trehalose Metabolism in Saccharomyces Cerevisiae from a Quantitative Approach

Saccharomyces cerevisiae utilizes two main mechanisms for storage of glucose: via glycogen and via trehalose. Though both pathways are closely related through the metabolism of G6P and UDPG, the onset of accumulation of these storage carbohydrates differs. It has been observed that this accumulation depends on growth conditions with glycogen accumulating mainly while the carbon source (i.e., glucose) is present in the medium. On the contrary, trehalose is usually formed when the glucose is close to depletion. At low growth rates S. cerevisiae accumulates high amounts of trehalose for two purposes: energy and carbon storage, and stabilization during stress conditions.

Trehalose can be converted into glucose through enzymatic hydrolysis catalyzed by either Ath1p or Nth1p/Nth2p. The former is known to operate on the outer cell surface (periplasm) converting trehalose extracellularly at acidic pH, while the latter is located in the cytosol and functions at neutral pH. Furthermore, the acidic Ath1p trehalase has been also found in the vacuole. Due to a continuous turnover that vary rapidly as a response to environmental changes and consumes ATP, it is expected that trehalose exhibits a high impact on the central metabolism, especially under dynamic conditions (e.g., at large industrial bioreactors or stimulus response experiments at bench-top scale) [1].

Although extensive information has been published on the role of trehalose in yeast metabolism, there is still few quantitative knowledge with respect to the extent of such an effect. For instance, flux estimations based on 13C-labeling experiments may be distorted due to the continuous cycling of trehalose. In order to elucidate the impact of trehalose metabolism from a quantitative point of view, we aim to quantify the dynamic response of the storage pools in relation with central metabolism under well-defined cultivation conditions. Thus, stable (¹³C) isotope labeling is used in combination with advance measurement techniques and modelling for non-stationary flux identification.

APPROACH

In this study, a wild type strain and two mutant strains deficient in trehalose metabolism were cultivated aerobically under chemostat (D = 0.1 h^-1) and feast/famine (dynamic cycles of high and low substrate availability) conditions. In addition U-13C-glucose was used as a tracer for later flux estimation by using measurements of mass isotopomers and concentrations. Thus, 13C-flux estimation has been performed at both steady and dynamic states.

During the metabolic dynamic experiment, the microorganisms were exposed to cyclic regimes of high and low substrate availability (feast/famine). In this case, the feast/famine consisted of an intermittent (block-wise) feeding regime in which the feeding pump was set on for 20s and then switch off for 380s making up a cycle time of 400s. During this cycle time the residual glucose concentration varied from high availability to almost complete depletion and vice versa. Highly reproducible cycles were obtained by the block-wise feeding allowing for extensive sampling for determination of metabolite concentrations and labeling state at different time-points during the cycle. The feast/famine setup may also be used for obtaining other types of samples (e.g., proteomics) which in combination with metabolite concentrations and labeling can be interpreted in the context of metabolic regulation and robustness.

RESULTS AND DISCUSSION

Clear and distinguishable dynamic responses to a perturbation were observed for the different pathways analyzed (upper and lower glycolysis, pentose phosphate, tricarboxylic acid and storage). In particular, 6PG and T6P exhibit delayed responses compared to their precursor G6P suggesting the presence of regulatory mechanisms. Estimation of both, the steady-state and feast/famine dynamic fluxes showed that most of the carbon flux at the G6P node was directed to glycolysis (about 80%), while about 10% was channeled to the pentose phosphate pathway and the rest was invested in the storage carbohydrate (trehalose and glycogen).

CONCLUSIONS

The aim of this work was to quantify up to what extent the metabolism of storage carbohydrates interacts with the central metabolism, and thus may alter the estimation of carbon fluxes. We determined that trehalose and glycogen can be recycled by diverting the carbon flux up to about 10-15% of the glucose uptake. The recycle of storage carbohydrates involves not only the consumption of energy (i.e., ATP) but also makes the estimation of carbon fluxes to be ill determined, especially during dynamic conditions. Finally, introducing an extension to the model of the central metabolism to account for storage recycle, revealed a significant flux through the trehalose node. In addition, 13C-labeling data proves to be highly informative in order to quantify these intracellular cycles.

[1] J. H. van Heerden, M. T. Wortel, F. J. Bruggeman, J. J. Heijnen, Y. J.M. Bollen, R. Planqué, J. Hulshof, T. G. O’Toole, S. A. Wahl,  B. Teusink.,  LOST IN TRANSITION: uncontrolled startup of glycolysis results in subpopulations of non-growing cells. Science (submitted), 2014