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Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. In this study, we clarified that a glucose response is involved in an inhibitory mechanism of proline utilization in yeast. Our genetic screen showed that strains with a loss-of-function mutation on the CDC25 gene can utilize proline even under fermentation conditions. Cdc25 is a regulator of the glucose response consisting of the Ras/cAMP-dependent protein kinase A (PKA) pathway. Moreover, we found that activation of the Ras/PKA pathway is necessary for the inhibitory mechanism of proline utilization. The present data revealed that crosstalk exists between the carbon and proline metabolisms. Our study could hold promise for the development of wine yeast strains that can efficiently assimilate proline during the fermentation processes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Proline is one of the abundant amino acids in grape must, but in winemaking processes it is poorly assimilated by the yeast Saccharomyces cerevisiae. This often causes a nitrogen deficiency during fermentation and proline accumulation in wine. Our previous study showed that arginine inhibits proline utilization by specifically inducing the endocytosis of the high-affinity proline transporter Put4. However, the detailed mechanisms underlying this induction are still unclear. Here, we propose a possible mechanism mediated by the ubiquitin ligase Rsp5 and its adaptor protein, Art3. First, we found that the ubiquitination activity of Rsp5 was essential for the arginine-induced endocytosis of Put4. Because Put4 contains no Rsp5-binding motif, we next screened an adaptor protein that plays a role in the arginine-induced endocytosis of Put4. Our genetic and biochemical analyses clearly revealed that the ART3 gene-disrupted cells were defective in Put4 endocytosis, indicating that Art3 is a key regulator for Put4 endocytosis. More importantly, we discovered that deletion of ART3 remarkably canceled the inhibitory effects of arginine on proline utilization. The present results could hold promise for the development of wine yeast strains that can efficiently assimilate the abundant proline in grape must during the fermentation processes., Competing Interests: Declaration of competing interest The authors (Akira Nishimura, Ryoya Tanahashi, and Hiroshi Takagi) declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality., (© 2020 John Wiley & Sons, Ltd.)

The proline metabolism intermediate Δ(1)-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The quality of alcoholic beverages strongly depends on the metabolic characteristics of the yeast cells being used. To control the aroma and the taste of alcoholic beverages, as well as the production of ethanol in them, it is thus crucial to select yeast cells with the proper characteristics. Grape must contain a high concentration of proline, an amino acid that can potentially be a useful nitrogen source. However, Saccharomyces cerevisiae cannot utilize proline during the wine-making process, resulting in the elevated levels of proline in wine and consequent negative effects on wine quality. In this article, I review and discuss recent discoveries about the inhibitory mechanisms and roles of proline utilization in yeast. The information can help in developing novel yeast strains that can improve fermentation and enhance the quality and production efficiency of wine. [ABSTRACT FROM AUTHOR]

Although proline is the most or second most abundant amino acid in wort and grape must, it is not fully consumed by the yeast Saccharomyces cerevisiae during alcoholic fermentation, unlike other amino acids. Our previous studies showed that arginine, the third most abundant amino acid in wort, inhibits the utilization of proline in most strains of S. cerevisiae. Furthermore, we found that some non-Saccharomyces yeasts utilized proline in a specific artificial medium with arginine and proline as the only nitrogen source, but these yeasts were not suitable for beer fermentation due to their low alcohol productivity. For yeasts to be useful for brewing, they need to utilize proline and produce alcohol during fermentation. In this study, 11 S. cerevisiae strains and 10 non-Saccharomyces yeast strains in the Phaff Yeast Culture Collection were identified that utilize proline effectively. Notably, two of these S. cerevisiae strains, UCDFST 40-144 and 68-44, utilize proline and produce sufficient alcohol in the beer fermentation model used. These strains have the potential to create distinctive beer products that are specifically alcoholic but with a reduction in proline in the finished beer. [ABSTRACT FROM AUTHOR]

Proline, which is a predominant amino acid in grape musts, is involved in the taste and flavor of foods and beverages. The yeast Saccharomyces cerevisiae poorly utilizes proline in wine-making processes, leading to a nitrogen deficiency during fermentation and proline accumulation in wine. Previous studies have shown that the protein kinase A (PKA) pathway is involved in inhibitory mechanisms of proline utilization. In this study, we screened the PKA pathway-related genes that regulate proline utilization. Using a yeast culture collection of disrupted strains associated with the downstream of the PKA cascade, we revealed that the stress-responsive transcription factor genes MSN2/4 regulate proline utilization. Moreover, we found that Msn2/4 up-regulate the SHY1 gene during the cell growth of the wine fermentation model, which may cause the inhibition of proline utilization. The SHY1 -deleted strain of the commercial wine yeast clearly showed proline consumption and average ethanol production under the wine fermentation model. The present data indicate that the PKA-Msn2/4-Shy1 cascade controls the inhibition of proline utilization under wine-making processes. Our study could hold promise for the development of wine yeast strains that can efficiently reduce proline during wine fermentation. [ABSTRACT FROM AUTHOR]

Abstract: The proline metabolism intermediate Δ1-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration. [Copyright &y& Elsevier]

Saccharomyces cerevisiaeΣ1278b has the MPR1 gene encoding the N-acetyltransferase Mpr1 that acetylates the proline metabolism intermediate Δ1-pyrroline-5-carboxylate (P5C)/glutamate-γ-semialdehyde (GSA) in vitro. In addition, Mpr1 protects cells from various oxidative stresses by regulating the levels of intracellular reactive oxygen species (ROS). However, the relationship between P5C/GSA acetylation and antioxidative mechanism involving Mpr1 remains unclear. Here, we report the synthesis of oxidative stress-induced arginine via P5C/GSA acetylation catalyzed by Mpr1. Gene disruption analysis revealed that Mpr1 converts P5C/GSA into N-acetyl-GSA for arginine synthesis in the mitochondria, indicating that Mpr1 mediates the proline and arginine metabolic pathways. More importantly, Mpr1 regulate ROS generation by acetylating toxic P5C/GSA. Under oxidative stress conditions, the transcription of PUT1 encoding the proline oxidase Put1 and MPR1 was strongly induced, and consequently, the arginine content was significantly increased. We also found that two deletion mutants (Δ mpr1/ 2 and Δ put1) were more sensitive to high-temperature stress than the wild-type strain, but that direct treatment with arginine restored the cell viability of these mutants. These results suggest that Mpr1-dependent arginine synthesis confers stress tolerance. We propose an antioxidative mechanism that is involved in stress-induced arginine synthesis requiring Mpr1 and Put1. [ABSTRACT FROM AUTHOR]

Awamori, the traditional distilled alcoholic beverage of Okinawa, Japan, is brewed with the yeast Saccharomyces cerevisiae. During the distillation process after the fermentation, enormous quantities of distillation residues containing yeast cells must be disposed of, and this has recently been recognized as a major problem both environmentally and economically. Proline, a multifunctional amino acid, has the highest water retention capacity among amino acids. Therefore, distillation residues with large amounts of proline could be useful in cosmetics. Here, we isolated a yeast mutant with high levels of intracellular proline and found a missense mutation (Gln79His) on the PRO1 gene encoding the γ-glutamyl kinase Pro1, a limiting enzyme in proline biosynthesis. The amino acid change of Gln79 to His in Pro1 resulted in desensitization to the proline-mediated feedback inhibition of GK activity, leading to the accumulation of proline in cells. Biochemical and in silico analyses showed that the amino acid residue at position 79 is involved in the stabilization of the proline binding pocket in Pro1 via a hydrogen-bonding network, which plays an important role in feedback inhibition. Our current study, therefore, proposed a possible mechanism underlying the feedback inhibition of γ-glutamyl kinase activity. This mechanism can be applied to construct proline-accumulating yeast strains to effectively utilize distillation residues. [ABSTRACT FROM AUTHOR]

Proline is a pivotal and multifunctional amino acid that is used not only as a nitrogen source but also as a stress protectant and energy source. Therefore, proline metabolism is known to be important in maintaining cellular homeostasis. Here, we discovered that proline oxidation, catalyzed by the proline oxidase Put1, a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate, controls the chronological lifespan of the yeast Saccharomyces cerevisiae. Intriguingly, the yeast strain with PUT1 deletion showed a reduced chronological lifespan compared with the wild-type strain. The addition of proline to the culture medium significantly increased the longevity of wild-type cells but not that of PUT1-deleted cells. We next found that induction of the transcriptional factor Put3-dependent PUT1 and degradation of proline occur during the aging of yeast cells. Additionally, the lifespan of the PUT3-deleted strain, which is deficient in PUT1 induction, was shorter than that of the wild-type strain. More importantly, the oxidation of proline by Put1 helped maintain the mitochondrial membrane potential and ATP production through the aging period. These results indicate that mitochondrial energy metabolism is maintained through oxidative degradation of proline and that this process is important in regulating the longevity of yeast cells. [ABSTRACT FROM AUTHOR]