Dataset Information

GTR1 Affects Nitrogen Consumption and TORC1 Activity in Saccharomyces cerevisiae Under Fermentation Conditions.

ABSTRACT: The TORC1 pathway coordinates cell growth in response to nitrogen availability present in the medium, regulating genes related to nitrogen transport and metabolism. Therefore, the adaptation of Saccharomyces cerevisiae to changes in nitrogen availability implies variations in the activity of this signaling pathway. In this sense, variations in nitrogen detection and signaling pathway are one of the main causes of differences in nitrogen assimilation during alcoholic fermentation. Previously, we demonstrated that allelic variants in the GTR1 gene underlying differences in ammonium and amino acids consumption between Wine/European (WE) and West African (WA) strains impact the expression of nitrogen transporters. The GTR1 gene encodes a GTPase that participates in the EGO complex responsible for TORC1 activation in response to amino acids availability. In this work, we assessed the role of the GTR1 gene on nitrogen consumption under fermentation conditions, using a high sugar concentration medium with nitrogen limitation and in the context of the WE and WA genetic backgrounds. The gtr1? mutant presented a reduced TORC1 activity and increased expression levels of nitrogen transporters, which in turn favored ammonium consumption, but decreased amino acid assimilation. Furthermore, to identify the SNPs responsible for differences in nitrogen consumption during alcoholic fermentation, we studied the polymorphisms present in the GTR1 gene. We carried out swapping experiments for the promoter and coding regions of GTR1 between the WE and WA strains. We observed that polymorphisms in the coding region of the WA GTR1 gene are relevant for TORC1 activity. Altogether, our results highlight the role of the GTR1 gene on nitrogen consumption in S. cerevisiae under fermentation conditions.

SUBMITTER: Molinet J

PROVIDER: S-EPMC7261904 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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GTR1 Affects Nitrogen Consumption and TORC1 Activity in Saccharomyces cerevisiae Under Fermentation Conditions.

Molinet Jennifer J Salinas Francisco F Guillamón José Manuel JM Martínez Claudio C

Frontiers in genetics 20200525

The TORC1 pathway coordinates cell growth in response to nitrogen availability present in the medium, regulating genes related to nitrogen transport and metabolism. Therefore, the adaptation of Saccharomyces cerevisiae to changes in nitrogen availability implies variations in the activity of this signaling pathway. In this sense, variations in nitrogen detection and signaling pathway are one of the main causes of differences in nitrogen assimilation during alcoholic fermentation. Previous ...[more]

PMID: 32523604

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Project description: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.

Project description:The eukaryotic domain-conserved TORC1 signalling pathway connects growth with nutrient sufficiency, promoting anabolic processes such as ribosomal biogenesis and protein synthesis. In Saccharomyces cerevisiae, TORC1 is activated mainly by the nitrogen sources. Recently, this pathway has gotten renewed attention but now in the context of the alcoholic fermentation, due to its key role in nitrogen metabolism regulation. Although the distal and proximal effectors downstream TORC1 are well characterised in yeast, the mechanism by which TORC1 is activated by nitrogen sources is not fully understood. In this work, we took advantage of a previously developed microculture-based methodology, which indirectly evaluates TORC1 activation in a nitrogen upshift experiment, to identify genetic variants affecting the activation of this pathway. We used this method to phenotype a recombinant population derived from two strains (SA and WE) with different geographic origins, which show opposite phenotypes for TORC1 activation by glutamine. Using this phenotypic information, we performed a QTL mapping that allowed us to identify several QTLs for TORC1 activation. Using a reciprocal hemizygous analysis, we validated GUS1, KAE1, PIB2, and UTH1 as genes responsible for the natural variation in the TORC1 activation. We observed that reciprocal hemizygous strains for KAE1 (ATPase required for t6A tRNA modification) gene showed the greatest phenotypic differences for TORC1 activation, with the hemizygous strain carrying the SA allele (KAE1 SA ) showing the higher TORC1 activation. In addition, we evaluated the fermentative capacities of the hemizygous strains under low nitrogen conditions, observing an antagonistic effect for KAE1 SA allele, where the hemizygous strain containing this allele presented the lower fermentation rate. Altogether, these results highlight the importance of the tRNA processing in TORC1 activation and connects this pathway with the yeasts fermentation kinetics under nitrogen-limited conditions.

Dataset Information

GTR1 Affects Nitrogen Consumption and TORC1 Activity in Saccharomyces cerevisiae Under Fermentation Conditions.

Publications

<i>GTR1</i> Affects Nitrogen Consumption and TORC1 Activity in <i>Saccharomyces cerevisiae</i> Under Fermentation Conditions.

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