scholarly journals Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease

2008 ◽  
Vol 74 (5) ◽  
pp. 1494-1501 ◽  
Author(s):  
Sergio L. Alves ◽  
Ricardo A. Herberts ◽  
Claudia Hollatz ◽  
Debora Trichez ◽  
Luiz C. Miletti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membranes ◽  
Yeast Cells ◽  
Sugar Uptake ◽  
Transport Activity ◽  
Brewing Industry ◽  
Sugar Consumption ◽  
Economic Problems ◽  
Yeast Strains ◽  
Kinetics Of

ABSTRACT Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known α-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (Km , 36 ± 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

scholarly journals PP2AB55δ Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Daisuke Watanabe ◽  
Takuma Kajihara ◽  
Yukiko Sugimoto ◽  
Kenichi Takagi ◽  
Megumi Mizuno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Alcoholic Fermentation ◽  
Yeast Cells ◽  
Loss Of Function ◽  
Fermentation Performance ◽  
Fermentation Rate ◽  
Yeast Strains ◽  
Evolutionarily Conserved ◽  
Fermentation Control

ABSTRACTSake yeast strain Kyokai no. 7 (K7) and its Saccharomyces cerevisiae relatives carry a homozygous loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-family protein kinase. Disruption of RIM15 in non-sake 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 B55Δ regulatory subunit (PP2AB55δ), 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 B55δ abolished the high fermentation performance of Rim15p-deficient laboratory yeast and sake yeast cells, indicating that PP2AB55δ mediates the fermentation control by TORC1 and Rim15p. The TORC1-Greatwall-PP2AB55δ pathway similarly affected the fermentation rate in the fission yeast Schizosaccharomyces pombe, strongly suggested that the evolutionarily conserved pathway governs alcoholic fermentation in yeasts. It is likely that elevated PP2AB55δ 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.IMPORTANCEThe 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-PP2AB55δ pathway plays a major role in the glycolytic control, our work may contribute to research on carbohydrate metabolism in higher eukaryotes.

scholarly journals A Loss-of-Function Mutation in the PAS Kinase Rim15p Is Related to Defective Quiescence Entry and High Fermentation Rates of Saccharomyces cerevisiae Sake Yeast Strains

2012 ◽  
Vol 78 (11) ◽  
pp. 4008-4016 ◽  
Author(s):  
Daisuke Watanabe ◽  
Yuya Araki ◽  
Yan Zhou ◽  
Naoki Maeya ◽  
Takeshi Akao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Frameshift Mutation ◽  
Laboratory Strain ◽  
Underlying Mechanism ◽  
Yeast Cells ◽  
Quiescent State ◽  
Loss Of Function ◽  
Content Type ◽  
Yeast Strains ◽  
Pas Kinase

ABSTRACTSake yeast cells have defective entry into the quiescent state, allowing them to sustain high fermentation rates. To reveal the underlying mechanism, we investigated the PAS kinase Rim15p, which orchestrates initiation of the quiescence program inSaccharomyces cerevisiae. We found that Rim15p is truncated at the carboxyl terminus in modern sake yeast strains as a result of a frameshift mutation. Introduction of this mutation or deletion of the full-lengthRIM15gene in a laboratory strain led to a defective stress response, decreased synthesis of the storage carbohydrates trehalose and glycogen, and impaired G1arrest, which together closely resemble the characteristic phenotypes of sake yeast. Notably, expression of a functionalRIM15gene in a modern sake strain suppressed all of these phenotypes, demonstrating that dysfunction of Rim15p prevents sake yeast cells from entering quiescence. Moreover, loss of Rim15p or its downstream targets Igo1p and Igo2p remarkably improved the fermentation rate in a laboratory strain. This finding verified that Rim15p-mediated entry into quiescence plays pivotal roles in the inhibition of ethanol fermentation. Taken together, our results suggest that the loss-of-function mutation in theRIM15gene may be the key genetic determinant of the increased ethanol production rates in modern sake yeast strains.

scholarly journals Partitioning of the 2-μm Circle Plasmid of Saccharomyces cerevisiae

2000 ◽  
Vol 149 (3) ◽  
pp. 553-566 ◽  
Author(s):  
Soundarapandian Velmurugan ◽  
Xian-Mei Yang ◽  
Clarence S.-M. Chan ◽  
Melanie Dobson ◽  
Makkuni Jayaram
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Cis Acting ◽  
Plasmid Segregation ◽  
Daughter Cells ◽  
Chromosomal Segregation ◽  
Rep Proteins ◽  
Cellular Machinery ◽  
2 Μm Plasmid ◽  
Kinetics Of

The efficient partitioning of the 2-μm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-μm–derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-μm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.

NADPH is important for isobutanol tolerance in a minimal medium of Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Yuki Yoshikawa ◽  
Ryo Nasuno ◽  
Hiroshi Takagi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Minimal Medium ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Acetaldehyde Dehydrogenase ◽  
Phosphogluconate Dehydrogenase ◽  
Yeast Strains ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract We showed that the isobutanol sensitivity in glucose-6-phosphate dehydrogenase-deficient cells of the yeast Saccharomyces cerevisiae was rescued by an alternative NADPH producer, acetaldehyde dehydrogenase, but not in the cells lacking 6-phosphogluconate dehydrogenase. This phenotype correlated with the intracellular NADPH/NADP+ ratio in yeast strains. Our findings indicate the importance of NADPH for the isobutanol tolerance of yeast cells.

scholarly journals Nutrient Signaling via the TORC1-Greatwall-PP2AB55δ Pathway Is Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
Daisuke Watanabe ◽  
Takuma Kajihara ◽  
Yukiko Sugimoto ◽  
Kenichi Takagi ◽  
Megumi Mizuno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Alcoholic Fermentation ◽  
Yeast Cells ◽  
Loss Of Function ◽  
Fermentation Performance ◽  
Content Type ◽  
Yeast Strains ◽  
Evolutionarily Conserved ◽  
Fermentation Control

ABSTRACT 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 B55δ regulatory subunit (PP2AB55δ), 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 B55δ, abolished the high fermentation performance of Rim15p-deficient laboratory yeast and sake yeast cells, indicating that PP2AB55δ mediates the fermentation control by TORC1 and Rim15p. The TORC1-Greatwall-PP2AB55δ 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 PP2AB55δ 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-PP2AB55δ pathway plays a major role in the glycolytic control, our work may contribute to research on carbohydrate metabolism in higher eukaryotes.

scholarly journals Effects of Crude β-Glucosidases from Issatchenkia terricola, Pichia kudriavzevii, Metschnikowia pulcherrima on the Flavor Complexity and Characteristics of Wines

2020 ◽  
Vol 8 (6) ◽  
pp. 953 ◽  
Author(s):  
Wenxia Zhang ◽  
Xuanhan Zhuo ◽  
Lanlan Hu ◽  
Xiuyan Zhang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Volatile Compound ◽  
Grape Juice ◽  
Physicochemical Characteristics ◽  
Sugar Consumption ◽  
Pichia Kudriavzevii ◽  
Metschnikowia Pulcherrima ◽  
Negative Effect ◽  
Kinetics Of

To investigate the effects of crude β-glucosidases from Issatchenkia terricola SLY-4 (SLY-4E), Pichia kudriavzevii F2-24 (F2-24E), and Metschnikowia pulcherrima HX-13 (HX-13E) on flavor complexity and characteristics of wines, grape juice was fermented by Saccharomyces cerevisiae with the addition of SLY-4E, F2-24E and HX-13E, respectively. The growth and sugar consumption kinetics of S. cerevisiae, the physicochemical characteristics, the volatile compounds, and the sensory dimensions of wines were analyzed. Results showed that adding SLY-4E, F2-24E, and HX-13E into must had no negative effect on the fermentation and physicochemical characteristics of wines, but increased the content of terpenes, esters, and fatty acids, while decreased the C6 compound content. Each wine had its typical volatile compound profiles. Adding SLY-4E or F2-24E into must could significantly improve the flavor complexity and characteristics of wines. These results would provide not only an approach to improve flavor complexity and characteristics of wines, but also references for application of β-glucosidases from other sources.

scholarly journals Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae

1996 ◽  
Vol 315 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Toshiko KASAHARA ◽  
Michihiro KASAHARA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Specific Activity ◽  
Expression System ◽  
Yeast Cells ◽  
Transport Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crude Membrane

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze–thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4±0.2 mM (mean±S.E.M.) and was inhibited by cytochalasin B (IC50 = 0.44±0.03 μM), HgCl2 (IC50 = 3.5±0.5 μM), phloretin (IC50 = 49±12 μM) and phloridzin (IC50 = 355±67 μM). To compare these properties with native GLUT1, we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.

Organic Acid Content, Antioxidant Capacity, and Fermentation Kinetics of Matured Coconut (Cocos nucifera) Water Fermented by Saccharomyces cerevisiae D254

2018 ◽  
Vol 14 (3) ◽  
Author(s):  
Guanfei Zhang ◽  
Xiaole Li ◽  
Wenxue Chen ◽  
Pusen Chen ◽  
Xiaofan Jin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Antioxidant Capacity ◽  
Cocos Nucifera ◽  
Coconut Water ◽  
Total Phenolic ◽  
Sugar Consumption ◽  
Yeast Growth ◽  
Alcohol Production ◽  
Kinetics Of

Abstract In this study, the quality of matured coconut water was improved through fermentation with Saccharomyces cerevisiae D254. During fermentation, the kinetic models of yeast growth, alcohol production, and sugar consumption were established based on logistic and Leudeking–Piret equations. Fructose, glucose, sucrose, total phenolic content, and antioxidant capacity (FRAP and ABTS values) were measured consecutively during fermentation. Results showed that R2 for the three models of yeast growth, alcohol production, and sugar consumption were 0.9772, 0.9983, and 0.9887, respectively. Total phenolic and antioxidant assays showed a similar evolution during fermentation, with a rapid increase in exponential phase and an unchanged trend in stationary phase. Moreover, total phenolic and the two antioxidant capacity methods were highly positively correlated. Pyruvic, lactic, citric, and succinic acids were the main organic acids in coconut water after fermentation.

scholarly journals Prm1 Targeting to Contact Sites Enhances Fusion during Mating in Saccharomyces cerevisiae

2010 ◽  
Vol 9 (10) ◽  
pp. 1538-1548 ◽  
Author(s):  
Valerie N. Olmo ◽  
Eric Grote
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Intracellular Transport ◽  
Plasma Membranes ◽  
Yeast Cells ◽  
Fusion Event ◽  
Mating Activity ◽  
Content Type ◽  
Contact Sites

ABSTRACT Prm1 is a pheromone-regulated membrane glycoprotein involved in the plasma membrane fusion event of Saccharomyces cerevisiae mating. Although this function suggests that Prm1 should act at contact sites in pairs of mating yeast cells where plasma membrane fusion occurs, only a small percentage of the total Prm1 was actually detected on the plasma membrane. We therefore investigated the intracellular transport of Prm1 and how this transport contributes to cell fusion. Two Prm1 chimeras that were sorted away from the contact site had reduced fusion activity, indicating that Prm1 indeed functions at contact sites. However, most Prm1 is located in endosomes and other cytoplasmic organelles and is targeted to vacuoles for degradation. Mutations in a putative endocytosis signal in a cytoplasmic loop partially stabilized the Prm1 protein and caused it to accumulate on the plasma membrane, but this endocytosis mutant actually had reduced mating activity. When Prm1 was expressed from a galactose-regulated promoter and its synthesis was repressed at the start of mating, vanishingly small amounts of Prm1 protein remained at the time when the plasma membranes came into contact. Nevertheless, this stable pool of Prm1 was retained at polarized sites on the plasma membrane and was sufficient to promote plasma membrane fusion. Thus, the amount of Prm1 expressed in mating yeast is far in excess of the amount required to facilitate fusion.

Germination studies on pure yeast ascospores

1974 ◽  
Vol 20 (11) ◽  
pp. 1517-1522 ◽  
Author(s):  
John J. Savarese
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Light Microscopy ◽  
Staining Technique ◽  
Sequential Treatment ◽  
Nutritional Requirements ◽  
Yeast Strains ◽  
Ascospore Germination ◽  
Kinetics Of ◽  
Acid Fast Staining

Pure ascospore suspensions of three strains of Saccharomyces cerevisiae have been prepared by sequential treatment with 2-mercaptoethanol, Glusulase, and sonication. Light microscopy and acid-fast staining revealed the presence of only ascospores. Various germination media tested using the ascospore preparation revealed different nutritional requirements for the germination of auxotrophic and prototrophic yeast strains. Acid-fast staining of the germinating ascospores was shown to be an effective method for studying the kinetics of ascospore germination. The anaerobic nature of ascospore germination was demonstrated using this staining technique.

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