Soybean Ferritin Expression in Saccharomyces cerevisiae Modulates Iron Accumulation and Resistance to Elevated Iron Concentrations

ABSTRACTFungi, including the yeastSaccharomyces cerevisiae, lack ferritin and use vacuoles as iron storage organelles. This work explored how plant ferritin expression influenced baker's yeast iron metabolism. Soybean seed ferritin H1 (SFerH1) and SFerH2 genes were cloned and expressed in yeast cells. Both soybean ferritins assembled as multimeric complexes, which bound yeast intracellular ironin vivoand, consequently, induced the activation of the genes expressed during iron scarcity. Soybean ferritin protected yeast cells that lacked the Ccc1 vacuolar iron detoxification transporter from toxic iron levels by reducing cellular oxidation, thus allowing growth at high iron concentrations. Interestingly, when simultaneously expressed inccc1Δ cells, SFerH1 and SFerH2 assembled as heteropolymers, which further increased iron resistance and reduced the oxidative stress produced by excess iron compared to ferritin homopolymer complexes. Finally, soybean ferritin expression led to increased iron accumulation in both wild-type andccc1Δ yeast cells at certain environmental iron concentrations.IMPORTANCEIron deficiency is a worldwide nutritional disorder to which women and children are especially vulnerable. A common strategy to combat iron deficiency consists of dietary supplementation with inorganic iron salts, whose bioavailability is very low. Iron-enriched yeasts and cereals are alternative strategies to diminish iron deficiency. Animals and plants possess large ferritin complexes that accumulate, detoxify, or buffer excess cellular iron. However, the yeastSaccharomyces cerevisiaelacks ferritin and uses vacuoles as iron storage organelles. Here, we explored how soybean ferritin expression influenced yeast iron metabolism, confirming that yeasts that express soybean seed ferritin could be explored as a novel strategy to increase dietary iron absorption.

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Responses of Saccharomyces cerevisiae Strains from Different Origins to Elevated Iron Concentrations

Applied and Environmental Microbiology ◽

10.1128/aem.03464-15 ◽

2016 ◽

Vol 82 (6) ◽

pp. 1906-1916 ◽

Cited By ~ 9

Author(s):

Carlos Andrés Martínez-Garay ◽

Rosa de Llanos ◽

Antonia María Romero ◽

María Teresa Martínez-Pastor ◽

Sergi Puig

Keyword(s):

Oxidative Stress ◽

Saccharomyces Cerevisiae ◽

Iron Deficiency ◽

Iron Concentration ◽

Yeast Cells ◽

Deficiency Anemia ◽

Content Type ◽

Yeast Strains ◽

Resistant Strains ◽

Iron Concentrations

ABSTRACTIron is an essential micronutrient for all eukaryotic organisms. However, the low solubility of ferric iron has tremendously increased the prevalence of iron deficiency anemia, especially in women and children, with dramatic consequences. Baker's yeastSaccharomyces cerevisiaeis used as a model eukaryotic organism, a fermentative microorganism, and a feed supplement. In this report, we explore the genetic diversity of 123 wild and domestic strains ofS. cerevisiaeisolated from different geographical origins and sources to characterize how yeast cells respond to elevated iron concentrations in the environment. By using two different forms of iron, we selected and characterized both iron-sensitive and iron-resistant yeast strains. We observed that when the iron concentration in the medium increases, iron-sensitive strains accumulate iron more rapidly than iron-resistant isolates. We observed that, consistent with excess iron leading to oxidative stress, the redox state of iron-sensitive strains was more oxidized than that of iron-resistant strains. Growth assays in the presence of different oxidative reagents ruled out that this phenotype was due to alterations in the general oxidative stress protection machinery. It was noteworthy that iron-resistant strains were more sensitive to iron deficiency conditions than iron-sensitive strains, which suggests that adaptation to either high or low iron is detrimental for the opposite condition. An initial gene expression analysis suggested that alterations in iron homeostasis genes could contribute to the different responses of distant iron-sensitive and iron-resistant yeast strains to elevated environmental iron levels.

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Determination of the Global Pattern of Gene Expression in Yeast Cells by Intracellular Levels of Guanine Nucleotides

mBio ◽

10.1128/mbio.02500-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Andy Hesketh ◽

Marta Vergnano ◽

Stephen G. Oliver

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Gene Transcription ◽

Adenine Nucleotide ◽

High Energy ◽

Yeast Cells ◽

Purine Nucleotides ◽

Global Pattern ◽

Content Type

ABSTRACT Correlations between gene transcription and the abundance of high-energy purine nucleotides in Saccharomyces cerevisiae have often been noted. However, there has been no systematic investigation of this phenomenon in the absence of confounding factors such as nutrient status and growth rate, and there is little hard evidence for a causal relationship. Whether transcription is fundamentally responsive to prevailing cellular energetic conditions via sensing of intracellular purine nucleotides, independently of specific nutrition, remains an important question. The controlled nutritional environment of chemostat culture revealed a strong correlation between ATP and GTP abundance and the transcription of genes required for growth. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into S. cerevisiae, permitting analysis of the transcriptional effect of an increased demand for these nucleotides. During steady-state growth using the fermentable carbon source glucose, the futile consumption of ATP led to a decrease in intracellular ATP concentration but an increase in GTP and the guanylate energy charge (GEC). Expression of transcripts encoding proteins involved in ribosome biogenesis, and those controlled by promoters subject to SWI/SNF-dependent chromatin remodelling, was correlated with these nucleotide pool changes. Similar nucleotide abundance changes were observed using a nonfermentable carbon source, but an effect on the growth-associated transcriptional programme was absent. Induction of the GTP-cycling pathway had only marginal effects on nucleotide abundance and gene transcription. The transcriptional response of respiring cells to glucose was dampened in chemostats induced for ATP cycling, but not GTP cycling, and this was primarily associated with altered adenine nucleotide levels. IMPORTANCE This paper investigates whether, independently of the supply of any specific nutrient, gene transcription responds to the energy status of the cell by monitoring ATP and GTP levels. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into the yeast Saccharomyces cerevisiae, and the effect of an increased demand for these purine nucleotides on gene transcription was analyzed. The resulting changes in transcription were most consistently associated with changes in GTP and GEC levels, although the reprogramming in gene expression during glucose repression is sensitive to adenine nucleotide levels. The results show that GTP levels play a central role in determining how genes act to respond to changes in energy supply and that any comprehensive understanding of the control of eukaryotic gene expression requires the elucidation of how changes in guanine nucleotide abundance are sensed and transduced to alter the global pattern of transcription.

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Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.00454-15 ◽

2015 ◽

Vol 81 (11) ◽

pp. 3688-3698 ◽

Cited By ~ 16

Author(s):

Kazutaka Sawada ◽

Tomoya Sato ◽

Hiroshi Hamajima ◽

Lahiru Niroshan Jayakody ◽

Miyo Hirata ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid Bacteria ◽

Yeast Cells ◽

Membrane Properties ◽

Yeast Culture ◽

Content Type ◽

Initial Stage ◽

Alkaline Conditions ◽

Metabolic Interactions ◽

Alkali Tolerance

ABSTRACTIn nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeastSaccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured withAspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer fromA. kawachiimodifies the physiology of the yeastS. cerevisiaeand demonstrate a new mechanism for cooperation between microbes in food fermentation.

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Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

Applied and Environmental Microbiology ◽

10.1128/aem.02838-19 ◽

2020 ◽

Vol 86 (7) ◽

Author(s):

Rui Yao ◽

Pei Zhou ◽

Chengjin Wu ◽

Liming Liu ◽

Jing Wu

Keyword(s):

Oxidative Stress ◽

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Survival Rate ◽

Type Strain ◽

Mutation Frequency ◽

Wild Type Strain ◽

Yeast Cells ◽

Wild Type ◽

Content Type

ABSTRACT In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

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Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.00027-17 ◽

2017 ◽

Vol 37 (11) ◽

Cited By ~ 26

Author(s):

Jayasankar Mohanakrishnan Kaimal ◽

Ganapathi Kandasamy ◽

Fabian Gasser ◽

Claes Andréasson

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Protein Aggregation ◽

Yeast Cells ◽

Eukaryotic Cells ◽

Content Type ◽

Dependent Protein ◽

And Function ◽

Cellular Dysfunction

ABSTRACT Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins.

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Sterol Composition Modulates the Response of Saccharomyces cerevisiae to Iron Deficiency

Journal of Fungi ◽

10.3390/jof7110901 ◽

2021 ◽

Vol 7 (11) ◽

pp. 901

Author(s):

Tania Jordá ◽

Nicolas Rozès ◽

Sergi Puig

Keyword(s):

Saccharomyces Cerevisiae ◽

Iron Deficiency ◽

Transcriptional Activation ◽

Fungal Infections ◽

Iron Acquisition ◽

Sterol Composition ◽

Yeast Cells ◽

Cellular Respiration ◽

Ergosterol Biosynthesis ◽

Yeast Saccharomyces Cerevisiae

Iron is a vital micronutrient that functions as an essential cofactor in multiple biological processes, including oxygen transport, cellular respiration, and metabolic pathways, such as sterol biosynthesis. However, its low bioavailability at physiological pH frequently leads to nutritional iron deficiency. The yeast Saccharomyces cerevisiae is extensively used to study iron and lipid metabolisms, as well as in multiple biotechnological applications. Despite iron being indispensable for yeast ergosterol biosynthesis and growth, little is known about their interconnections. Here, we used lipid composition analyses to determine that changes in the pattern of sterols impair the response to iron deprivation of yeast cells. Yeast mutants defective in ergosterol biosynthesis display defects in the transcriptional activation of the iron-acquisition machinery and growth defects in iron-depleted conditions. The transcriptional activation function of the iron-sensing Aft1 factor is interrupted due to its mislocalization to the vacuole. These data uncover novel links between iron and sterol metabolisms that need to be considered when producing yeast-derived foods or when treating fungal infections with drugs that target the ergosterol biosynthesis pathway.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.00165-12 ◽

2012 ◽

Vol 78 (11) ◽

pp. 4008-4016 ◽

Cited By ~ 54

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.

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Pho85 Kinase, a Cyclin-Dependent Kinase, Regulates Nuclear Accumulation of the Rim101 Transcription Factor in the Stress Response of Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00247-09 ◽

2010 ◽

Vol 9 (6) ◽

pp. 943-951 ◽

Cited By ~ 13

Author(s):

Masafumi Nishizawa ◽

Mirai Tanigawa ◽

Michio Hayashi ◽

Tatsuya Maeda ◽

Yoshiaki Yazaki ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Cellular Response ◽

Proteolytic Processing ◽

Yeast Cells ◽

Nuclear Accumulation ◽

Alkaline Stress ◽

Content Type ◽

Its Gene ◽

Alkaline Conditions

ABSTRACT The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither RIM101 transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that PTK2 activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the PHO system when yeast cells cope with various environmental stresses potentially threatening their survival.

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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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Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase

Applied and Environmental Microbiology ◽

10.1128/aem.03717-15 ◽

2016 ◽

Vol 82 (6) ◽

pp. 1706-1721 ◽

Cited By ~ 8

Author(s):

Eduard Calam ◽

Eva González-Roca ◽

M. Rosario Fernández ◽

Sylvie Dequin ◽

Xavier Parés ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Protein ◽

Formate Dehydrogenase ◽

Building Blocks ◽

Yeast Cells ◽

Content Type ◽

Permeabilized Cells ◽

Oxidation Reduction ◽

New Variant ◽

Butanediol Dehydrogenase

ABSTRACTButanediol dehydrogenase (Bdh1p) fromSaccharomyces cerevisiaebelongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversiblyR-acetoin andS-acetoin to (2R,3R)-2,3-butanediol andmeso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of thedelitto perfettomethodology to placeBDH1under the control of theGAL1promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.

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