Management of Multiple Nitrogen Sources during Wine Fermentation by Saccharomyces cerevisiae

Abstract

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 ␣-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.KEYWORDS complex nitrogen resource, metabolic network, metabolism, nitrogen, quantitative analysis, regulation, yeasts T he management of nutrients provided by the external environment, mainly carbon and nitrogen, and their redistribution inside the cells for the formation of biosynthetic precursors through the metabolic network are essential for all living organisms. The topology of the metabolic network of the yeast Saccharomyces cerevisiae is one of

A comparison of the nitrogen metabolic networks of Kluyveromyces marxianus and Saccharomyces cerevisiae

“…C). As previously observed for S. cerevisiae (Crépin et al ., ), the amount of arginine taken up greatly exceeded the amount directly incorporated into proteins (Fig. C, Table S2, Data Set S5).…”

“…With this aim, the fraction of these three amino acids present in biomass which originated directly from the exogenous compound was quantified. As already observed for valine and leucine in S. cerevisiae (Crépin et al ., ; Rollero et al ., ), a substantial imbalance was observed in the early growth phase between the level of consumption of the exogenous amino acids and the content of these compounds in proteins (Fig. A, Fig A and B, Table S3) in K. marxianus .…”

“…In all fermentations, all nitrogen sources were provided, but only one was 15 N‐ (ammonium, arginine and glutamine) or 13 C‐ (leucine, valine and phenylalanine) labelled. The former three amino acids were chosen as they represent the most abundant (almost 70% of the nitrogen resource) and the preferred sources of nitrogen of S. cerevisiae in winemaking conditions (Crépin, 2012; Crépin et al ., ) and their redistribution within the de novo proteinogenic amino acids was assessed. The latter three were studied because they are direct precursors of the most common higher alcohols found in wine that are, at least partially, produced through the Ehrlich pathway (Ehrlich, ; Hazelwood et al ., ).…”

“…To determine the origin of nitrogen for de novo amino acid synthesis throughout the fermentation, the redistribution of nitrogen from arginine, glutamine and ammonium to proteinogenic amino acids (except for cysteine, glutamine, methionine, tryptophan and tyrosine, which were not detected by the GC–MS method used) was explored during fermentation in the presence of 15 N‐labelled sources. As previously observed for S. cerevisiae (Crépin et al ., ), a substantial portion of the proteinogenic amino acids were synthesized de novo using the nitrogen originating from these three sources (Fig. B, Table S2, Data Set S5).…”

13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched‐chain amino acids

“…Newly synthesized protein contains Glu with a reduced fraction of intact C α –C β bonds because a full cycle of the TCA cycle yields C α –C β bonds in which the two carbon atoms originate from two source molecules of glucose. In contrast, Pro degradation (a two reaction pathway) to Glu is suppressed in the presence of ammonium in S. cerevisiae . Like for Glu, Asp can be interconverted to OAA through transamination and enters the TCA cycle.…”