Identification of informative metabolic responses using a minibioreactor: a small step change in the glucose supply rate creates a large metabolic response in Saccharomyces cerevisiae

Abstract

In this study, a previously developed mini-bioreactor, the Biocurve, was used to identify an informative stimulus-response experiment. The identified stimulus-response experiment was a modest 50% shift-up in glucose uptake rate (qGLC) that unexpectedly resulted in a disproportionate transient metabolic response. The 50% shift-up in qGLC in the Biocurve resulted in a near tripling of the online measured oxygen uptake (qO 2 ) and carbon dioxide production (qCO 2 ) rates, suggesting a considerable mobilization of glycogen and trehalose. The 50% shift-up in qGLC was subsequently studied in detail in a conventional bioreactor (4 l working volume), which confirmed the results obtained with the Biocurve. Especially relevant is the observation that the 50% increase in glucose uptake rate led to a three-fold increase in glycolytic flux, due to mobilization of storage materials. This explains the unexpected ethanol and acetate secretion after the shift-up, in spite of the fact that after the shift-up the qGLC was far less than the critical value. Moreover, these results show that the correct in vivo fluxes in glucose pulse experiments cannot be obtained from the uptake and secretion rates only. Instead, the storage fluxes must also be accurately quantified. Finally, we speculate on the possible role that the transient increase in dissolved CO 2 immediately after the 50% shift-up in qGLC could have played a part in triggering glycogen and trehalose mobilization.

Quantification of the transient and long-term response of Saccharomyces cerevisiae to carbon dioxide stresses of various intensities

“…Total biomass corresponds to the sum of the active biomass, trehalose and glycogen. The total biomass formation rate was calculated as the mean value of the two values calculated using the two balances [15]. Both values showed excellent agreement (data not shown).…”

“…Therefore the transient total biomass formation rate was calculated from the carbon and degree of reduction balances as described by [15,37]. Total biomass corresponds to the sum of the active biomass, trehalose and glycogen.…”

“…Some studies conducted in tightly controlled conditions and/or using synthetic media are nevertheless available [8,[15][16][17][18][19]. The reduction in the biomass yield reached 54% for a CO 2,l concentration of 25.45 mM in an aerobic carbon-limited fed-batch culture [18] and 24% for a CO 2,l concentration of 22.30 mM in an aerobic carbon-limited chemostat culture [8].…”

“…Carbon dioxide influence on yeast metabolism has been studied in fermentative metabolism [6][7][8][9][10], oxido-reductive metabolism [8,[10][11][12][13][14] and oxidative metabolism [8,[15][16][17][18]. Biomass growth, fermentative capacity and in some case product formation are affected by CO 2 although this impact varies greatly depending on the strain, on the cultivation mode (batch, fed-batch, continuous) and on the yeast metabolism.…”

“…Higher specific respiration rates (+74%) were also observed in the latter case. The short-term response of yeast to CO 2 has been studied by a stimulus-response strategy that revealed that a step increase of CO 2,l concentration triggered a transient increase of the respiratory activity linked to the mobilization of storage sugars [15][16][17].…”