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Genetic variants of TORC1 signaling pathway affect nitrogen consumption in Saccharomyces cerevisiae during alcoholic fermentation.

ABSTRACT: In the alcoholic fermentation process, Saccharomyces cerevisiae strains present differences in their nitrogen consumption profiles, these phenotypic outcomes have complex genetic and molecular architectures. In this sense, variations in nitrogen signaling pathways regulated by TORC1 represent one of the main sources of phenotypic diversity in nitrogen consumption. This emphasizes the possible roles that allelic variants from the TORC1 pathway have in the nitrogen consumption differences observed in yeast during the alcoholic fermentation. Here, we studied the allelic diversity in the TORC1 pathway across four yeast strains and determined how these polymorphisms directly impact nitrogen consumption during alcoholic fermentation. Using a reciprocal hemizygosity approach combined with phenotyping under fermentative conditions, we found that allelic variants of GTR1, TOR2, SIT4, SAP185, EAP1, NPR1 and SCH9 underlie differences in the ammonium and amino acids consumption phenotypes. Among these, GTR1 alleles from the Wine/European and West African genetic backgrounds showed the greatest effects on ammonium and amino acid consumption, respectively. Furthermore, we identified allelic variants of SAP185, TOR2, SCH9 and NPR1 from an oak isolate that increased the amino acid consumption preference over ammonium; representing putative candidates coming from a non-domesticated strain that could be used for genetic improvement programs. In conclusion, our results demonstrated that a large number of allelic variants within the TORC1 pathway significantly impacts on regulatory mechanisms of nitrogen assimilation during alcoholic fermentation.

SUBMITTER: Molinet J

PROVIDER: S-EPMC6660096 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 (K7) and its relatives carry a homozygous loss-of-function mutation in the RIM15 gene, which encodes a Greatwall family protein kinase. Disruption of RIM15 in nonsake yeast strains leads to improved alcoholic fermentation, indicating that the defect in Rim15p is associated with the enhanced fermentation performance of sake yeast cells. In order to understand how Rim15p mediates fermentation control, we here focused on target-of-rapamycin protein kinase complex 1 (TORC1) and protein phosphatase 2A with the B55? regulatory subunit (PP2AB55?), complexes that are known to act upstream and downstream of Rim15p, respectively. Several lines of evidence, including our previous transcriptomic analysis data, suggested enhanced TORC1 signaling in sake yeast cells during sake fermentation. Fermentation tests of the TORC1-related mutants using a laboratory strain revealed that TORC1 signaling positively regulates the initial fermentation rate in a Rim15p-dependent manner. Deletion of the CDC55 gene, encoding B55?, abolished the high fermentation performance of Rim15p-deficient laboratory yeast and sake yeast cells, indicating that PP2AB55? mediates the fermentation control by TORC1 and Rim15p. The TORC1-Greatwall-PP2AB55? pathway similarly affected the fermentation rate in the fission yeast Schizosaccharomyces pombe, strongly suggesting that the evolutionarily conserved pathway governs alcoholic fermentation in yeasts. It is likely that elevated PP2AB55? activity accounts for the high fermentation performance of sake yeast cells. Heterozygous loss-of-function mutations in CDC55 found in K7-related sake strains may indicate that the Rim15p-deficient phenotypes are disadvantageous to cell survival.IMPORTANCE The biochemical processes and enzymes responsible for glycolysis and alcoholic fermentation by the yeast S. cerevisiae have long been the subject of scientific research. Nevertheless, the factors determining fermentation performance in vivo are not fully understood. As a result, the industrial breeding of yeast strains has required empirical characterization of fermentation by screening numerous mutants through laborious fermentation tests. To establish a rational and efficient breeding strategy, key regulators of alcoholic fermentation need to be identified. In the present study, we focused on how sake yeast strains of S. cerevisiae have acquired high alcoholic fermentation performance. Our findings provide a rational molecular basis to design yeast strains with optimal fermentation performance for production of alcoholic beverages and bioethanol. In addition, as the evolutionarily conserved TORC1-Greatwall-PP2AB55? pathway plays a major role in the glycolytic control, our work may contribute to research on carbohydrate metabolism in higher eukaryotes.

Project description:BACKGROUND: The expression and activity of the different Saccharomyces cerevisiae hexose uptake systems (Hxt) and the kinetics of glucose uptake are considered essential to industrial alcoholic fermentation performance. However, the dynamics of glucose uptake kinetics during the different stages of fermentation, depending on glucose and nitrogen availability, is very poorly characterized. The objective of the present work was to examine thoroughly the alterations occurring in glucose uptake kinetics during alcoholic fermentation, by the wine strain S. cerevisiae PYCC 4072, of a synthetic grape juice basal medium with either a limiting or non-limiting initial nitrogen concentration and following nitrogen supplementation of the nitrogen-depleted sluggish fermentation. RESULTS: Independently of the initial concentration of the nitrogen source, glucose transport capacity is maximal during the early stages of fermentation and presumably sustained by the low-affinity and high-capacity glucose transporter Hxt1p. During nitrogen-limited sluggish fermentation, glucose uptake capacity was reduced to approximately 20% of its initial values (Vmax?=?4.9?±?0.8 compared to 21.9?±?1.2??mol?h?¹ 10?? cells), being presumably sustained by the low-affinity glucose transporter Hxt3p (considering the calculated Km?=?39.2?±?8.6?mM). The supplementation of the sluggish fermentation broth with ammonium led to the increase of glucose transport capacity associated to the expression of different glucose uptake systems with low and high affinities for glucose (Km?=?58.2?±?9.1 and 2.7?±?0.4?mM). A biclustering analysis carried out using microarray data, previously obtained for this yeast strain transcriptional response to equivalent fermentation conditions, indicates that the activation of the expression of genes encoding the glucose transporters Hxt2p (during the transition period to active fermentation) and Hxt3p, Hxt4p, Hxt6p and Hxt7p (during the period of active fermentation) may have a major role in the recovery of glucose uptake rate following ammonium supplementation. These results suggest a general derepression of the glucose-repressible HXT genes and are consistent with the downregulation of Mig1p and Rgt1p. CONCLUSIONS: Although reduced, glucose uptake rate during nitrogen-limited fermentation is not abrogated. Following ammonium supplementation, sluggish fermentation recovery is associated to the increase of glucose uptake capacity, related to the de novo synthesis of glucose transporters with different affinity for glucose and capacity, presumably of Hxt2p, Hxt3p, Hxt4p, Hxt6p and Hxt7p. This study is a contribution to the understanding of yeast response to different stages of alcoholic fermentation at the level of glucose uptake kinetics, in particular under nitrogen limitation or replenish, which is useful knowledge to guide fermentation practices.

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Genetic variants of TORC1 signaling pathway affect nitrogen consumption in Saccharomyces cerevisiae during alcoholic fermentation.

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