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During fermentative growth in natural and industrial environments, Saccharomyces cerevisiae must redistribute the available nitrogen from multiple exogenous sources to amino acids in order to suitably fulfill anabolic requirements. To exhaustively explore the management of this complex resource, we developed an advanced strategy based on the reconciliation of data from a set of stable isotope tracer experiments with labeled nitrogen sources. Thus, quantifying the partitioning of the N compounds through the metabolism network during fermentation, we demonstrated that, contrary to the generally accepted view, only a limited fraction of most of the consumed amino acids is directly incorporated into proteins. Moreover, substantial catabolism of these molecules allows for efficient redistribution of nitrogen, supporting the operative de novo synthesis of proteinogenic amino acids. In contrast, catabolism of consumed amino acids plays a minor role in the formation of volatile compounds. Another important feature is that the α-keto acid precursors required for the de novo syntheses originate mainly from the catabolism of sugars, with a limited contribution from the anabolism of consumed amino acids. This work provides a comprehensive view of the intracellular fate of consumed nitrogen sources and the metabolic origin of proteinogenic amino acids, highlighting a strategy of distribution of metabolic fluxes implemented by yeast as a means of adapting to environments with changing and scarce nitrogen resources. IMPORTANCE A current challenge for the wine industry, in view of the extensive competition in the worldwide market, is to meet consumer expectations regarding the sensory profile of the product while ensuring an efficient fermentation process. Understanding the intracellular fate of the nitrogen sources available in grape juice is essential to the achievement of these objectives, since nitrogen utilization affects both the fermentative activity of yeasts and the formation of flavor compounds. However, little is known about how the metabolism operates when nitrogen is provided as a composite mixture, as in grape must. Here we quantitatively describe the distribution through the yeast metabolic network of the N moieties and C backbones of these nitrogen sources. Knowledge about the management of a complex resource, which is devoted to improvement of the use of the scarce N nutrient for growth, will be useful for better control of the fermentation process and the sensory quality of wines., (Copyright © 2017 American Society for Microbiology.)

The efficiency of nitrogen use is a key determinant of the completion of alcoholic fermentation. We analyzed the kinetics of consumption of 18 nitrogen compounds by 14 Saccharomyces cerevisiae strains of various origins in a synthetic medium that mimicked a grape must. The kinetic profiles of total nitrogen consumption were diverse, but the order of nitrogen source consumption was similar for all strains. The nitrogen compounds could be classified into three groups, according to their order of use: prematurely consumed (Lys), early consumed (Asp, Thr, Glu, Leu, His, Met, Ile, Ser, Gln, and Phe), and late consumed (ammonium, Val, Arg, Ala, Trp, and Tyr). The initial concentrations of these compounds did not alter the order in which they were consumed, except for arginine and ammonium. Early consumed amino acids are transported by specific permeases under Ssy1p-Ptr3p-Ssy5 (SPS)-mediated control that are expressed at the beginning of consumption. Most nitrogen compounds consumed late are transported by permeases under nitrogen catabolite repression (NCR), and others (Val, Trp, and Tyr) are transported by SPS-regulated low-affinity permeases. Therefore, the kinetic characteristics of transporters, as well as SPS and NCR, are likely key factors controlling the temporal sequence of consumption of nitrogen compounds and constitute a system highly conserved in S. cerevisiae species. This work sheds new light on the mechanistic basis of the sequential use of different nitrogen compounds in complex environments.