Involvement of nitrogen metabolism in the triggering of ethanol fermentation in aerobic chemostat cultures of Saccharomyces cerevisiae

We have investigated whether central nitrogen metabolism may influence the triggering of ethanol fermentation in Saccharomyces cerevisiae strain CEN.PK122 grown in the presence of different N-sources (ammonia, glutamate, or glutamine) under conditions in which the carbon to nitrogen (C:N) ratio was varied. An exhaustive quantitative evaluation of yeast physiology and metabolic behavior through metabolic flux analysis (MFA) was undertaken. It is shown that ethanol fermentation is triggered at dilution rates, D (growth rate), significantly lower (D = 0.070 and 0.074 h^-1 for glutamate and glutamine, respectively, and D = 0.109 h^-1 for ammonia) under N- than C-limitation ( ∼ 0.18 h^-1 for all N-sources). A characteristic specific rate of glucose influx, q_Glc, for each N-source at Dc, i.e., just before the onset of respirofermentative metabolism, was determined ( ∼ 2.0, 1.5, and 2.5, for ammonia, glutamate, and glutamine, respectively). This q_Glc was independent of the nutritional limitation though dependent on the nature of the N-source. The onset of fermentation occurs when this "threshold q_Glc" is overcome. The saturation of respiratory activity appears not to be associated with the onset of fermentation since q_O2 continued to increase after Dc. It was remarkable that under respirofermentative conditions in C-limited chemostat cultures, the glucose consumed was almost completely fermented with biomass being synthesized from glutamate through gluconeogenesis. The results obtained show that the enzyme activities involved in central nitrogen metabolism do not appear to participate in the control of the overflow in carbon catabolism, which is driven toward ethanol production. The role of nitrogen metabolism in the onset of ethanol fermentation would rather be realized through its involvement in setting the anabolic fluxes directed to nitrogenous macromolecules. It seems that nitrogen-related anabolic fluxes would determine when the threshold glucose consumption rate is achieved after which ethanol fermentation is triggered.