Saccharomyces cerevisiae yeast immobilized on marrow stem sunflower and polyacrylamide hydrogels

AbstractBiocatalysts with microorganisms immobilized on solid carriers could provide the solution for development of continuous industrial processes for ethanol obtaining by fermentation of sugars. In this study, modified polyacrylamide hydrogels and marrow stem sunflower are used as supports for Saccharomyces cerevisiae yeast immobilization. The obtained structures are used for fermentation of molasses in batch systems. The free yeast cells are used as reference. The modification of polyacrilamide matrix with (2-hydroxyethyl)methacrylate has a positive effect on structure pore uniformity and fermentation performance. The mechanical properties of the obtained biocatalysts are compared. The novel natural matrix has net superior compression strength.

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

10.1101/402081 ◽

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.

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Understanding the Impact of Industrial Stress Conditions on Replicative Aging in Saccharomyces cerevisiae

Frontiers in Fungal Biology ◽

10.3389/ffunb.2021.665490 ◽

2021 ◽

Vol 2 ◽

Author(s):

Marco Eigenfeld ◽

Roland Kerpes ◽

Thomas Becker

Keyword(s):

Saccharomyces Cerevisiae ◽

Real Time ◽

High Throughput ◽

Stress Conditions ◽

Yeast Cells ◽

Industrial Processes ◽

Non Invasive ◽

Age Related ◽

Yeast Aging ◽

The Impact

In yeast, aging is widely understood as the decline of physiological function and the decreasing ability to adapt to environmental changes. Saccharomyces cerevisiae has become an important model organism for the investigation of these processes. Yeast is used in industrial processes (beer and wine production), and several stress conditions can influence its intracellular aging processes. The aim of this review is to summarize the current knowledge on applied stress conditions, such as osmotic pressure, primary metabolites (e.g., ethanol), low pH, oxidative stress, heat on aging indicators, age-related physiological changes, and yeast longevity. There is clear evidence that yeast cells are exposed to many stressors influencing viability and vitality, leading to an age-related shift in age distribution. Currently, there is a lack of rapid, non-invasive methods allowing the investigation of aspects of yeast aging in real time on a single-cell basis using the high-throughput approach. Methods such as micromanipulation, centrifugal elutriator, or biotinylation do not provide real-time information on age distributions in industrial processes. In contrast, innovative approaches, such as non-invasive fluorescence coupled flow cytometry intended for high-throughput measurements, could be promising for determining the replicative age of yeast cells in fermentation and its impact on industrial stress conditions.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.02083-18 ◽

2018 ◽

Vol 85 (1) ◽

Cited By ~ 2

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.

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Immobilization of Saccharomyces cerevisiae on Apple Pieces to Produce Cider

Fermentation ◽

10.3390/fermentation5030074 ◽

2019 ◽

Vol 5 (3) ◽

pp. 74 ◽

Cited By ~ 1

Author(s):

Clelia Altieri ◽

Daniela Campaniello ◽

Barbara Speranza ◽

Milena Sinigaglia ◽

Maria Rosaria Corbo ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Viable Count ◽

Second Step ◽

Small Scale ◽

Refrigerated Storage ◽

Fermentation Performance ◽

Wild Yeast ◽

Free Cells ◽

Probiotic Yeast ◽

Yeast Immobilization

Three yeasts (Saccharomyces cerevisiae var. boulardii, a commercial probiotic yeast; S. cerevisiae W13, a wild yeast able to remove ochratoxin A; and S. cerevisiae 17, a wild yeast with promising probiotic traits) were screened for their ability to adhere on apple pieces as a function of different contact times (15–30 min). Then, apple pieces were stored at 4 °C for 15 days, and the viable count of yeasts was periodically assessed. Yeasts were able to adhere on apple pieces after 15 min (7 log cfu/g) and retained their viability throughout the refrigerated storage. In a second step, apple pieces with S. cerevisiae W13 were used to produce cider on a small scale. The variables under investigation were (a) the recycling of pieces up to 10 times and (b) the preliminary storage of pieces at 4 °C before use. Pieces used immediately after yeast immobilization could be successfully used again 10 times and gained a fermentation performance (in terms of yeast amount in cider and ethanol after 24 h) similar to that achieved by free cells. In addition, the preliminary storage of pieces at 4 °C did not affect their performances as reusable starter carriers.

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The effect of preconditioning cells under osmotic stress on high alcohol production

Zbornik Matice srpske za prirodne nauke ◽

10.2298/zmspn1324405l ◽

2013 ◽

pp. 405-414 ◽

Cited By ~ 1

Author(s):

Stilijanos Logotetis ◽

Panagiotis Tataridis ◽

Anastasios Kanelis ◽

Elijas Nerancis

Keyword(s):

Saccharomyces Cerevisiae ◽

Osmotic Stress ◽

Yeast Cells ◽

High Alcohol ◽

Fermentation Performance ◽

Yeast Saccharomyces Cerevisiae ◽

Simple Method ◽

Alcohol Production ◽

Fermentation Processes ◽

Ethanol Toxicity

This paper focuses on the research into the influence of salt on physiology of the yeast, Saccharomyces cerevisiae. Specifically, the work focused on how NaCl affected the growth, viability and fermentation performance of this yeast in laboratory-scale experiments. One of the main findings of the research presented involved the influ?ence of salt ?preconditioning? of yeasts which represents a method of pre-culturing of cells in the presence of salt in an attempt to improve subsequent fermentation performance. Such an approach resulted in preconditioned yeasts having an improved capability to ferment high-sugar containing media (up to 60% w/v of glucose) with increased cell viability and with increased levels of produced ethanol (higher than 20% in vol.). Salt-preconditioning was most likely influencing the stress-tolerance of yeasts by inducing the synthesis of key metabolites such as trehalose and glycerol which act to improve cells? ability to withstand osmostress and ethanol toxicity. The industrial-scale trials using salt-preconditioned yeasts verified the benefit of the physiological engineering approach to practical fermentations. Overall, this research has demonstrated that a relatively simple method designed to adapt yeast cells physiologically - by salt-preconditioning - can have distinct advantages for al?cohol fermentation processes.

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Eukaryotic translation factor eIF5A contributes to acetic acid tolerance in Saccharomyces cerevisiae via transcriptional factor Ume6p

Biotechnology for Biofuels ◽

10.1186/s13068-021-01885-2 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yanfei Cheng ◽

Hui Zhu ◽

Zhengda Du ◽

Xuena Guo ◽

Chenyao Zhou ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid ◽

Adaptive Response ◽

Acid Tolerance ◽

Peptide Bond ◽

Transcriptional Factor ◽

Yeast Cells ◽

Acetic Acid Tolerance ◽

Translation Factor ◽

Industrial Strains

Abstract Background Saccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown. Results In this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance. Conclusion Translation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.

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The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/166.4.1687 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1687-1699

Author(s):

Jesús de la Cruz ◽

Thierry Lacombe ◽

Olivier Deloche ◽

Patrick Linder ◽

Dieter Kressler

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosome Biogenesis ◽

Null Allele ◽

Ribosomal Subunit ◽

Interaction Network ◽

The Novel ◽

Rna Helicases ◽

Ribosomal Subunits ◽

Synthetic Lethal ◽

Synthetically Lethal

Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

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Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Genes ◽

10.3390/genes12020219 ◽

2021 ◽

Vol 12 (2) ◽

pp. 219

Author(s):

Il-Sup Kim ◽

Woong Choi ◽

Jonghyeon Son ◽

Jun Hyuck Lee ◽

Hyoungseok Lee ◽

...

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Transcription Factors ◽

Stress Tolerance ◽

Genetic Network ◽

Yeast Cells ◽

Enzymatic Antioxidants ◽

Freezing And Thawing ◽

Metal Ion Homeostasis ◽

Transgenic Yeast

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

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Pulsed Electric Field (PEF) Enhances Iron Uptake by the Yeast Saccharomyces cerevisiae

Biomolecules ◽

10.3390/biom11060850 ◽

2021 ◽

Vol 11 (6) ◽

pp. 850

Author(s):

Karolina Nowosad ◽

Monika Sujka ◽

Urszula Pankiewicz ◽

Damijan Miklavčič ◽

Marta Arczewska

Keyword(s):

Saccharomyces Cerevisiae ◽

Electric Field ◽

Metal Ion ◽

Treatment Time ◽

Control Sample ◽

Pulsed Electric Field ◽

Yeast Cells ◽

Iron Ions ◽

Metal Ion Binding ◽

Yeast Saccharomyces Cerevisiae

The aim of the study was to investigate the influence of a pulsed electric field (PEF) on the level of iron ion accumulation in Saccharomyces cerevisiae cells and to select PEF conditions optimal for the highest uptake of this element. Iron ions were accumulated most efficiently when their source was iron (III) nitrate. When the following conditions of PEF treatment were used: voltage 1500 V, pulse width 10 μs, treatment time 20 min, and a number of pulses 1200, accumulation of iron ions in the cells from a 20 h-culture reached a maximum value of 48.01 mg/g dry mass. Application of the optimal PEF conditions thus increased iron accumulation in cells by 157% as compared to the sample enriched with iron without PEF. The second derivative of the FTIR spectra of iron-loaded and -unloaded yeast cells allowed us to determine the functional groups which may be involved in metal ion binding. The exposure of cells to PEF treatment only slightly influenced the biomass and cell viability. However, iron-enriched yeast (both with or without PEF) showed lower fermentative activity than a control sample. Thus obtained yeast biomass containing a high amount of incorporated iron may serve as an alternative to pharmacological supplementation in the state of iron deficiency.

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Improving Ethyl Acetate Production in Baijiu Manufacture by Wickerhamomyces anomalus and Saccharomyces cerevisiae Mixed Culture Fermentations

BioMed Research International ◽

10.1155/2019/1470543 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Guangsen Fan ◽

Chao Teng ◽

Dai Xu ◽

Zhilei Fu ◽

Pengxiao Liu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethyl Acetate ◽

Mixed Culture ◽

Nutrient Competition ◽

Acetate Production ◽

Mixed Fermentation ◽

Wickerhamomyces Anomalus ◽

Flavor Perception ◽

Positive Effect

Ethyl acetate content has strong influence on the style and quality of Baijiu. Therefore, this study investigated the effect of Saccharomyces cerevisiae Y3401 on the production of ethyl acetate by Wickerhamomyces anomalus Y3604. Analysis of cell growth showed that Y3401 influences Y3604 by nutrient competition and inhibition by metabolites, while the effect of Y3604 on Y3401 was mainly competition for nutrients. Mixed fermentation with two yeasts was found to produce more ethyl acetate than a single fermentation. The highest yield of ethyl acetate was 2.99 g/L when the inoculation ratio of Y3401:Y3604 was 1:2. Synergistic fermentation of both yeasts improved ethyl acetate production and increased the content of other flavor compounds in liquid and simulated solid-state fermentation for Baijiu. Saccharomyces cerevisiae had a positive effect on ethyl acetate production in mixed culture and provides opportunities to alter the aroma and flavor perception of Baijiu.

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