Threonine Overproduction in Yeast Strains Carrying theHOM3-R2 Mutant Allele under the Control of Different Inducible Promoters

ABSTRACT The HOM3 gene of Saccharomyces cerevisiaecodes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis. With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the HOM3-R2 mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, P GAL1 , P CHA1 , P CYC1-HSE2 , and P GPH1 . The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions. Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed. Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation. Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors.

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Differentiation of Yeast Cultures in Ruminant Feedingstuffs Using a Rapid DNA Fingerprinting Technique

Proceedings of the British Society of Animal Production (1972) ◽

10.1017/s0308229600026489 ◽

1994 ◽

Vol 1994 ◽

pp. 101-101

Author(s):

Elizabeth Moore ◽

Denis R. Headon

Keyword(s):

Sodium Dodecyl ◽

Yeast Cells ◽

Microbial Populations ◽

Yeast Culture ◽

Yeast Cultures ◽

Yeast Strains ◽

Genetic Sequences ◽

Polymerase Chain ◽

Different Strains ◽

Triton X

Research indicates that certain yeast strains are beneficial in their capacity to stimulate key microbial populations. This stimulation is strain specific with similar yeast strains exerting their effect on totally different microbial populations. Future yeast culture supplements may contain mixtures of different strains designed to suit specific diets. This, therefore, requires the development of a rapid sensitive technique to differentiate among taxonomically similar yeast strains in animal diets. This technique, termed the Randomly Amplified Polymorphic DNA (RAPD) assay, is based upon the use of randomly designed short polynucleotide primers to amplify genetic sequences from the DNA of the desired yeast strain. Our objective involves the development of this technique to distinguish between closely related yeast strains present in feed. The feed sample investigated was a standard cattle ration containing three strains of Saccharomyces cerevisiae (1026, 2045 and 2020) and Candida utilis 3001 at a concentration of 106 CFU/g respectively. Isolation of single colonies of yeast strains present was achieved by feed extraction in dilution buffer followed by plating a series of dilutions on rose-bengal agar. Thirty randomly selected colonies were cultured in YPD (1% yeast extract, 2% peptone, 2% glucose) broth for 24 - 30 hours at 30°C. Genomic DNA was isolated from yeast cells by standard methods based on subjection of the cells to vortex mixing in the presence of glass beads, triton X-100, sodium dodecyl sulphate, phenol and chloroform. Isolated DNA from randomly selected colonies was amplified by Polymerase Chain Reaction (PCR) for 45 cycles of 1 min at 94°C, 1 min at 36°C and 1 min at 72°C using randomly designed 10 bp primers.

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The Quality of Ciders Depends on the Must Supplementation with Mineral Salts

Molecules ◽

10.3390/molecules25163640 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3640

Author(s):

Tomasz Tarko ◽

Magdalena Januszek ◽

Aneta Pater ◽

Paweł Sroka ◽

Aleksandra Duda-Chodak

Keyword(s):

Antioxidant Properties ◽

Control Sample ◽

Yeast Cells ◽

Polyphenol Content ◽

Mineral Salts ◽

Total Polyphenol ◽

Total Polyphenol Content ◽

Yeast Strains ◽

Magnesium Salts ◽

The Impact

Providing yeast with the right amount of mineral salts before fermentation can contribute to improving the entire technological process, resulting in a better-quality final product. The aim of this study was to assess the impact of apple must supplementation with mineral salts ((NH4)2SO4, MgSO4, (NH4)3PO4)) on enological parameters, antioxidant activity, total polyphenol content, and the profile of volatile cider compounds fermented with various yeast strains. Rubin cultivar must was inoculated with wine, cider, and distillery or wild yeast strains. Various mineral salts and their mixtures were introduced into the must in doses from 0.167 g/L to 0.5 g/L. The control sample consisted of ciders with no added mineral salts. The basic enological parameters, antioxidant properties, total polyphenol content, and their profile, as well as the composition of volatile compounds, were assessed in ciders. Must supplementation with magnesium salts significantly influenced the use of the analyzed element by yeast cells and was dependent on the yeast strain. In supplemented samples, a decrease in alcohol concentration and total acidity, as well as an increase in the content of extract and total polyphenols, was observed compared to the controls. The addition of ammonium salts caused a decrease in the amount of higher alcohols and magnesium salts, as well as a decrease in the concentration of some esters in ciders.

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Reproductive Potential of Yeast Cells Depends on Overall Action of Interconnected Changes in Central Carbon Metabolism, Cellular Biosynthetic Capacity, and Proteostasis

International Journal of Molecular Sciences ◽

10.3390/ijms21197313 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7313

Author(s):

Roman Maslanka ◽

Renata Zadrag-Tecza

Keyword(s):

Calorie Restriction ◽

Carbon Metabolism ◽

Metabolic Flux ◽

Reproductive Potential ◽

Central Carbon Metabolism ◽

Yeast Cells ◽

Reproductive Capacity ◽

Hexokinase 2 ◽

Central Carbon

Carbon metabolism is a crucial aspect of cell life. Glucose, as the primary source of energy and carbon skeleton, determines the type of cell metabolism and biosynthetic capabilities, which, through the regulation of cell size, may affect the reproductive capacity of the yeast cell. Calorie restriction is considered as the most effective way to improve cellular physiological capacity, and its molecular mechanisms are complex and include several nutrient signaling pathways. It is widely assumed that the metabolic shift from fermentation to respiration is treated as a substantial driving force for the mechanism of calorie restriction and its influence on reproductive capabilities of cells. In this paper, we propose another approach to this issue based on analysis the connection between energy-producing and biomass formation pathways which are closed in the metabolic triangle, i.e., the respiration-glycolysis-pentose phosphate pathway. The analyses were based on the use of cells lacking hexokinase 2 (∆hxk2) and conditions of different glucose concentration corresponding to the calorie restriction and the calorie excess. Hexokinase 2 is the key enzyme involved in central carbon metabolism and is also treated as a calorie restriction mimetic. The experimental model used allows us to explain both the role of increased respiration as an effect of calorie restriction but also other aspects of carbon metabolism and the related metabolic flux in regulation of reproductive potential of the cells. The obtained results reveal that increased respiration is not a prerequisite for reproductive potential extension but rather an accompanying effect of the positive role of calorie restriction. More important seems to be the changes connected with fluxes in central carbon metabolic pathways resulting in low biosynthetic capabilities and improved proteostasis.

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Diversity of marine yeasts with high protein content and evaluation of their nutritive compositions

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315408001938 ◽

2008 ◽

Vol 88 (7) ◽

pp. 1347-1352 ◽

Cited By ~ 12

Author(s):

Zhenming Chi ◽

Kuiran Yan ◽

Lingmei Gao ◽

Jing Li ◽

Xianghong Wang ◽

...

Keyword(s):

Amino Acids ◽

Fatty Acids ◽

Nucleic Acid ◽

Dry Weight ◽

Essential Amino Acids ◽

Marine Yeasts ◽

Marine Animals ◽

Hanseniaspora Uvarum ◽

Yeast Strains ◽

Routine Identification

A total of 327 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish and marine algae were obtained. After crude protein of the yeasts was estimated by the method of Kjehldahl, we found that eight strains of the marine yeasts grown in the medium with 20 g l−1 glucose contained more than 30.4 g protein per 100 g of cell dry weight. The results of routine identification and molecular methods show that they belong to Metschnikowa reukaui, Cryptococcus aureus, Aureobasidium pullulan, Yarrowia lipolytica and Hanseniaspora uvarum, respectively. With the exception of Aureobasidium pullulans 4#2 with nucleic acid of 7.7% (w/w), all other yeast strains contained less than 5.0% (w/w) of nucleic acid. Analysis of fatty acids shows that all the yeast strains tested had a large amount of C18:0 and C18:1 fatty acids while analysis of amino acids indicates that the yeast strains tested had a large amount of essential amino acids, especially lysine and leucine which are very important nutritive components for marine animals.

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Isolation and Characterization of Live Yeast Cells from Ancient Vessels as a Tool in Bio-Archaeology

mBio ◽

10.1128/mbio.00388-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 10

Author(s):

Tzemach Aouizerat ◽

Itai Gutman ◽

Yitzhak Paz ◽

Aren M. Maeir ◽

Yuval Gadot ◽

...

Keyword(s):

Ancient Dna ◽

Residue Analysis ◽

Fermented Food ◽

Yeast Cells ◽

Fermented Beverages ◽

Isolation And Characterization ◽

Yeast Strains ◽

Novel Approach ◽

Clay Matrix

ABSTRACTAncient fermented food has been studied based on recipes, residue analysis, and ancient-DNA techniques and reconstructed using modern domesticated yeast. Here, we present a novel approach based on our hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. We developed a pipeline of yeast isolation from clay vessels and screened for yeast cells in beverage-related and non-beverage-related ancient vessels and sediments from several archaeological sites. We found that yeast cells could be successfully isolated specifically from clay containers of fermented beverages. The findings that genotypically the isolated yeasts are similar to those found in traditional African beverages and phenotypically they grow similar to modern beer-producing yeast strongly suggest that they are descendants of the original fermenting yeast. These results demonstrate that modern microorganisms can serve as a new tool in bio-archaeology research.IMPORTANCESo far, most of the study of ancient organisms has been based mainly on the analysis of ancient DNA. Here we show that it is possible to isolate and study microorganisms—yeast in this case—from ancient pottery vessels used for fermentation. We demonstrate that it is highly likely that these cells are descendants of the original yeast strains that participated in the fermentation process and were absorbed into the clay matrix of the pottery vessels. Moreover, we characterized the isolated yeast strains, their genomes, and the beer they produced. These results open new and exciting avenues in the study of domesticated microorganisms and contribute significantly to the fields of bio- and experimental archaeology that aim to reconstruct ancient artifacts and products.

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Accumulation of Selenium in Candida utilis Growing in Media of Increasing Concentration of this Element

Applied Sciences ◽

10.3390/app10041439 ◽

2020 ◽

Vol 10 (4) ◽

pp. 1439 ◽

Cited By ~ 3

Author(s):

Marek Kieliszek ◽

Anna Maria Kot ◽

Kamil Piwowarek ◽

Stanisław Błażejak

Keyword(s):

Candida Utilis ◽

Culture Medium ◽

Dry Weight ◽

Yeast Cells ◽

Reciprocating Motion ◽

Experimental Medium ◽

Yeast Cultures ◽

Cell Biomass ◽

Icp Ms ◽

Living Organisms

Selenium is considered an essential component of all living organisms. Studies on the enrichment of yeast cells with selenium, using the ability of cell biomass to bind this element, are being reported more and more. Yeast cultures were cultivated in YPD medium enriched with Na2SeO3 salts for 72 h at 28 °C on a shaker utilizing reciprocating motion. Selenium in cell biomass was determined with the use of ICP–MS. It was observed that the addition of selenium to the experimental medium (in the range of 4–100 mg/L) increased the content of this element in the yeast cell biomass. During the extension of cultivation time, the number of yeast cells and biomass yield exhibited a decreasing trend. Based on the obtained results, it was concluded that yeast cells exhibited the ability to accumulate selenium in both logarithmic and stationary growth phases. The dose of 20 and 30 mg/L of selenium in the culture medium meets the expectations in terms of both the content of selenium bound to yeast cells (1944 ± 110.8 μg/g dry weight) under 48-h cultivation. The obtained results confirmed that the Candida utilis ATCC 9950 strain exhibits the ability to bind selenium, which means that the biomass of these yeasts may be used as a natural source of selenium in the diet of humans and animals.

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Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses

Applied and Environmental Microbiology ◽

10.1128/aem.00376-16 ◽

2016 ◽

Vol 82 (10) ◽

pp. 3121-3130 ◽

Cited By ~ 23

Author(s):

Sirikarn Charoenbhakdi ◽

Thanittra Dokpikul ◽

Thanawat Burphan ◽

Todsapol Techo ◽

Choowong Auesukaree

Keyword(s):

Cell Wall ◽

Alcoholic Fermentation ◽

Ethanol Tolerance ◽

Wall Stress ◽

Yeast Cells ◽

Intracellular Acidification ◽

Straight Chain ◽

Cell Wall Stress ◽

Yeast Strains ◽

Vacuolar Acidification

ABSTRACTDuring fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H+-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2and Δvma3mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype ofvmamutants resulted from severe cytosolic acidification. Interestingly, thevmamutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol.IMPORTANCEThe yeastSaccharomyces cerevisiaehas been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic fermentation. The results revealed that straight-chain alcohols induced cytosolic and vacuolar acidification through their membrane-permeabilizing effects. Contrary to expectations, a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress, but not in the maintenance of intracellular pH, seems to be important for protecting yeast cells against ethanol stress. These findings will expand our understanding of the mechanisms of ethanol tolerance and provide promising clues for the development of ethanol-tolerant yeast strains.

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Genotoxicity of Chemical Compounds Identification and Assessment by Yeast Cells Transformed With GFP Reporter Constructs Regulated by the PLM2 or DIN7 Promoter

International Journal of Toxicology ◽

10.1177/1091581814566870 ◽

2015 ◽

Vol 34 (1) ◽

pp. 31-43 ◽

Cited By ~ 6

Author(s):

Van Ngoc Bui ◽

Thi Thu Huyen Nguyen ◽

Yvan Bettarel ◽

Thi Hoai Thu Nguyen ◽

Thuy Linh Pham ◽

...

Keyword(s):

Dna Damage ◽

High Throughput Screening ◽

Fluorescent Protein ◽

Chemical Compounds ◽

Cost Effective ◽

Yeast Cells ◽

Fluorescence Signal ◽

Inducible Promoters ◽

Dna Damaging Agents ◽

Green Fluorescent

Yeast cells transformed with high-copy number plasmids comprising a green fluorescent protein (GFP)-encoding gene optimized for yeast under the control of the new DIN7 or PLM2 and the established RNR2 and RAD54 promoters were used to assess the genotoxic potential of chemical compounds. The activity of potential DNA-damaging agents was investigated by genotoxicity assays and by OxoPlate assay in the presence of various test compounds. The fluorescence signal generated by GFP in response to DNA damage was related to the different concentrations of analytes and the analyte-dependent GFP synthesis. The use of distinct DNA damage-inducible promoters presents alternative genotoxicity testing strategies by selective induction of promoters in response to DNA damage. The new DIN7 and PLM2 systems show higher sensitivity than the RNR2 and RAD54 systems in detecting 4-nitroquinoline- N-oxide and actinomycin D. Both DIN7 and PLM2 systems are able to detect camptothecin while RNR2 and RAD54 systems are not. Automated laboratory systems with assay performance on 384-well microplates provide for cost-effective high-throughput screening of DNA-damaging agents, reducing compound consumption to about 53% as compared with existing eukaryotic genotoxicity bioassays.

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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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Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

10.1101/682302 ◽

2019 ◽

Author(s):

Francisca Monteiro ◽

Georg Hubmann ◽

Justin Norder ◽

Johan Hekelaar ◽

Joana Saldida ◽

...

Keyword(s):

Protein Design ◽

Metabolic Flux ◽

Single Cells ◽

Yeast Cells ◽

Glycolytic Flux ◽

Cell Level ◽

Yeast Cultures ◽

Intrinsic Correlation ◽

Metabolic Heterogeneity ◽

Fructose 1,6 Bisphosphate

AbstractMetabolic heterogeneity between individual cells of a population harbors offers significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence requires tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e. the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells, by drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), and by transplanting the B. subtilis FBP-binding transcription factor CggR into yeast. As proof of principle, using fluorescence microscopy, we applied the sensor to identify metabolic subpopulations in yeast cultures. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

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