Volatile compounds produced by yeast during fermentation greatly influence the organoleptic qualities of wine. We developed a model to predict the combined effects of initial nitrogen and phytosterol content and fermentation temperature on the production of volatile compounds. We used a Box-Behnken design and response surface modeling to study the response of Lalvin EC1118® to these environmental conditions. Initial nitrogen content had the greatest influence on most compounds; however, there were differences in the value of fermentation parameters required for the maximal production of the various compounds. Fermentation parameters affected differently the production of isobutanol and isoamyl alcohol, although their synthesis involve the same enzymes and intermediate. We found differences in regulation of the synthesis of acetates of higher alcohols and ethyl esters, suggesting that fatty acid availability is the main factor influencing the synthesis of ethyl esters whereas the production of acetates depends on the activity of alcohol acetyltransferases. We also evaluated the effect of temperature on the total production of three esters by determining gas-liquid balances. Evaporation largely accounted for the effect of temperature on the accumulation of esters in liquid. Nonetheless, the metabolism of isoamyl acetate and ethyl octanoate was significantly affected by this parameter. We extended this study to other strains. Environmental parameters had a similar effect on aroma production in most strains. Nevertheless, the regulation of the synthesis of fermentative aromas was atypical in two strains: Lalvin K1M® and Affinity™ ECA5, which produces a high amount of aromatic compounds and was obtained by experimental evolution.

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

The sequential inoculation of non-Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species (Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae, that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae. The metabolic footprint of the non-Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae. For instance, fermentations performed with K. marxianus were characterized by the formation of phenylethanol and phenylethyl acetate, while those performed with P. burtonii or Z. meyerae displayed higher production of isoamyl alcohol and ethyl esters. When considering sequential inoculation of yeasts, the nutritional requirements of the yeasts used should be carefully considered and adjusted accordingly. Finally, our chemical data suggests that the organoleptic properties of the wine are altered in a species specific manner.