Effect of S-adenosyl-methionine accumulation on hineka odor in sake brewed with a non-Kyokai yeast.

Hineka is a type of off-flavor of sake and is attributed to the presence of several compounds, including a major one called dimethyl trisulfide (DMTS). The production of the main precursor of DMTS involves yeast methionine salvage pathway. The DMTS-producing potential (DMTS-pp) of sake brewed using the Km67 strain, a non-Kyokai sake yeast, is lower than that of sake brewed using Kyokai yeast; however, the detailed mechanism is unclear. We focused on S-adenosyl-methionine (SAM) and aimed to elucidate the mechanism that prevents DMTS production in sake brewed using the Km67 strain. We revealed that SAM is involved in DMTS production in sake, and that the conversion of SAM to the DMTS precursor occurs through an enzymatic reaction rather than a chemical reaction. Based on previous reports on ADO1 and MDE1 genes, sake brewing tests were performed using the Km67 Δmde1, Δado1, and Δmde1Δado1 strains. A comparison of the SAM content of pressed sake cakes and DMTS-pp of sake produced using the Km67 Δado1 strain showed an increase in both SAM content and DMTS-pp compared to those produced using the parent strain. However, the Km67 Δmde1Δado1 strain showed little increase in DMTS-pp compared to the Km67 Δmde1 strain, despite an increase in SAM content. These results suggest that SAM accumulation in yeast plays a role in the production of DMTS in sake through the methionine salvage pathway. Moreover, the low SAM-accumulation characteristic of the Km67 strain contributes to low DMTS production in sake.

Importance and mechanisms of S-adenosylmethionine and folate accumulation in sake yeast.

Sake yeasts have a range of brewing characteristics that are particularly beneficial for sake making including high ethanol fermentability, high proliferative capacity at low temperatures, lactic acid tolerance, and high ester productivity. On the other hand, sake yeasts also accumulate a diverse range of functional components. For example, significantly greater accumulation of S-adenosylmethionine (SAM), a compound that plays important regulatory roles in a range of biological processes as a major donor of methyl groups, occurs in sake yeasts compared to other microorganisms. Significantly greater accumulation of folate, a bioactive water-soluble vitamin (vitamin B9), also occurs in sake yeasts compared to laboratory yeasts, and the methyl group on SAM is supplied by folate. Accordingly, fully characterizing 'sake yeast identity' requires detailed understanding of the mechanisms underlying both the nutritional characteristics (functional components) and the brewing characteristics in sake yeasts. Therefore, this mini-review focuses on the accumulation of SAM and folate in sake yeast including descriptions of the genes known to contribute to SAM and folate accumulation and the underlying mechanisms.

S-adenosyl-L-homocysteine extends lifespan through methionine restriction effects.

Methionine restriction (MetR) can extend lifespan and delay the onset of aging-associated pathologies in most model organisms. Previously, we showed that supplementation with the metabolite S-adenosyl-L-homocysteine (SAH) extends lifespan and activates the energy sensor AMP-activated protein kinase (AMPK) in the budding yeast Saccharomyces cerevisiae. However, the mechanism involved and whether SAH can extend metazoan lifespan have remained unknown. Here, we show that SAH supplementation reduces Met levels and recapitulates many physiological and molecular effects of MetR. In yeast, SAH supplementation leads to inhibition of the target of rapamycin complex 1 (TORC1) and activation of autophagy. Furthermore, in Caenorhabditis elegans SAH treatment extends lifespan by activating AMPK and providing benefits of MetR. Therefore, we propose that SAH can be used as an intervention to lower intracellular Met and confer benefits of MetR.

Quantitative stability of the folates highly accumulated in a non-Kyokai sake yeast.

Pressed sake cake, a by-product of sake brewing, is a rich dietary source of folates, which are important vitamins for humans. However, considerable losses of folates occur during storage and cooking. We have previously reported that Km67, the house sake yeast strain of Kiku-masamune sake brewery, can accumulate high folate levels. In this study, we found that the folate content of pressed sake cakes produced with Km67 remained at approximately their maximum level after the fermentation activity stopped. To elucidate the mechanisms of high folate accumulation in Km67, we analyzed the expression of 23 folate-metabolizing genes. The expression of ABZ1 and FOL3 was almost always higher in Km67 than in Kyokai no. 701 yeast (K701), which suggested that enhanced expression of the genes involved in folate biosynthesis was a mechanism of high folate accumulation in Km67. We found that the folates of Km67 pressed sake cakes were quantitatively stable at 4°C under refrigerated storage conditions. In addition, the homocysteine content of Km67 pressed sake cakes was almost always higher than that of K701 pressed sake cakes. This result suggests that a reason for high folate accumulation in Km67 yeast is the need to reduce the intracellular concentration of homocysteine. Our results provide biologically meaningful information on folate metabolism in yeast.

Development of sake yeast haploid set with diverse brewing properties using sake yeast strain Hiroshima no. 6 exhibiting sexual reproduction.

Among sake yeast strains, Kyokai no. 7 (K7) and its closely related strains (K7 group) are predominantly used because of their excellent brewing properties. In the sake industrial sector, the need for various types of yeast strains is high. Although crossbreeding is an effective method for generating genetic diversity that should result in diverse characteristics, most K7 group strains lack normal sporulation ability, including the ability to undergo meiotic chromosomal recombination, which leads to difficulties in crossbreeding. Accordingly, the improvement of sake yeast strains primarily depends on mutagenesis and suitable selection in a stepwise manner. Our recent study revealed that the long-preserved sake yeast strain Hiroshima no. 6 (H6) does not belong to the K7 group despite genetically being extremely similar. In addition, H6 exhibited normal sporulation. Thus, we isolated haploid cells from H6 and mated them with previously isolated haploid cells of K7 group strains. The crossbred diploid strains had normal sporulation ability; hence, we performed tetrad analysis. The brewing characteristics of the obtained haploid set were extremely diverse. Principal component analysis based on the volatile and organic acid components measured using small-scale sake brewing tests revealed that the haploid strains derived from each diploid strain displayed a characteristic distribution. Thus, we demonstrated the availability of genetic crossbreeding using H6 with sporulation ability to facilitate both the development of novel sake yeast strains with many desirable characteristics and analyses of the function of sake yeast.

The sake yeast YHR032W/ERC1 allele contributes to the regulation of the tetrahydrofolate content in the folate synthetic pathway in sake yeast strains.

To elucidate the mechanism underlying tetrahydrofolate (THF) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed a quantitative trait locus (QTL) analysis. The results revealed that the sake yeast ERC1 allele contributes to an increase in the ratio of THF to the total folate content in sake yeast.

SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae.

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.

Breeding of a cordycepin-resistant and adenosine kinase-deficient sake yeast strain that accumulates high levels of S-adenosylmethionine.

Adenosine kinase (ADO1)-deficient mutants can be obtained from cordycepin-resistant strains, and the disruption of ADO1 causes S-adenosylmethionine (SAM) accumulation. To breed a high-SAM-accumulating yeast strain without genetic manipulation for industrial purposes, we bred a cordycepin-resistant strain using sake yeast kyokai No. 9 as the parent strain with a mutation in adenosine kinase (ADO1) and acquired high-SAM-accumulating strain. In the bred strain (NY9-10), a single mutation (T258I) was present in the ADO1, and this mutation site is an ATP binding site and is highly conserved during evolution. Moreover, it was suggested that high accumulation of SAM and cordycepin resistance in NY9-10 was due to functional deficiency of ADO1 by this mutation. This strain is not a genetically-modified organism and can be employed for use in the food and medicine industry such as mass production and sake making.

A genetic method to enhance the accumulation of S-adenosylmethionine in yeast.

S-Adenosylmethionine (SAM) is a key component of sulphur amino acid metabolism in living organisms and is synthesised from methionine and adenosine triphosphate by methionine adenosyltransferase. This molecule serves as the main biological methyl donor due to its active methylthio ether group. Notably, SAM has shown beneficial effects in clinical trials for the treatment of alcoholic liver disease, depression and joint pain. Due to the high potential value of SAM, current research efforts are attempting to develop a more rapid, cost-effective and higher yielding SAM production method than the conventional production system. In this mini-review, we describe the previously reported yeast gene that contributes to SAM accumulation by overexpression, mutation or deletion and summarise the genetic approach for the production of SAM in large industrial quantities.

Sake yeast YHR032W/ERC1 haplotype contributes to high S-adenosylmethionine accumulation in sake yeast strains.

Sake yeasts are ideally suited for sake making, producing higher levels of ethanol, proliferating at lower temperatures, and producing greater levels of various aromatic components and nutrients than laboratory yeasts. To elucidate the mechanism underlying S-adenosylmethionine (SAM) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed quantitative trait locus (QTL) analysis and identified a significant QTL on chromosome VIII. Of the 165 genes mapped at 49.8 cM from the left-end DNA marker of chromosome VIII, we focused on the YHR032W/ERC1 gene, encoding a member of the multi-drug and toxin extrusion family having antiporter activity and involved in SAM accumulation and ethionine resistance. Expression of the sake yeast ERC1 haplotype (K7ERC1) in a low- and high-copy number plasmid BYΔerc1 resulted in intracellular SAM accumulation, whereas expression of the laboratory yeast ERC1 haplotype (XERC1) did not. Comparison between DNA sequences of K7ERC1 and XERC1 revealed three amino acid substitutions: S51N, V263I, and N545I. Site-directed mutagenesis revealed that the N545I frameshift mutation was responsible for the K7ERC1 phenotype. These results indicate that K7ERC1 contributes to SAM accumulation in sake yeast strains.

Stimulating S-adenosyl-l-methionine synthesis extends lifespan via activation of AMPK.

Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-l-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-l-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.

The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts.

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature-sensitive fission yeast strains, rrs1-D14/22G and rrs1-L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1-84 (D22/30G) and rrs1-124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two-hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts.

RSC Chromatin-Remodeling Complex Is Important for Mitochondrial Function in Saccharomyces cerevisiae.

RSC (Remodel the Structure of Chromatin) is an ATP-dependent chromatin remodeling complex essential for the growth of Saccharomyces cerevisiae. RSC exists as two distinct isoforms that share core subunits including the ATPase subunit Nps1/Sth1 but contain either Rsc1or Rsc2. Using the synthetic genetic array (SGA) of the non-essential null mutation method, we screened for mutations exhibiting synthetic growth defects in combination with the temperature-sensitive mutant, nps1-105, and found connections between mitochondrial function and RSC. rsc mutants, including rsc1Δ, rsc2Δ, and nps1-13, another temperature-sensitive nps1 mutant, exhibited defective respiratory growth; in addition, rsc2Δ and nps1-13 contained aggregated mitochondria. The rsc2Δ phenotypes were relieved by RSC1 overexpression, indicating that the isoforms play a redundant role in respiratory growth. Genome-wide expression analysis in nps1-13 under respiratory conditions suggested that RSC regulates the transcription of some target genes of the HAP complex, a transcriptional activator of respiratory gene expression. Nps1 physically interacted with Hap4, the transcriptional activator moiety of the HAP complex, and overexpression of HAP4 alleviated respiratory defects in nps1-13, suggesting that RSC plays pivotal roles in mitochondrial gene expression and shares a set of target genes with the HAP complex.

Yeast cell lysis enhances dimethyl trisulfide formation in sake.

The present study showed that the lysis of yeast cells and subsequent release of cell contents in sake mash accelerated dimethyl trisulfide (DMTS) formation. Among these, heat unstable and relatively high molecular weight compounds were assumed to be enzymes; thus, enzymatic reactions probably contribute to DMTS formation.

Statistical analysis of sake-preparation conditions and dimethyl trisulfide formation.

Dimethyl trisulfide (DMTS) is known to be responsible for hineka, an off-flavor that develops during storage, in sake. Previous studies have attempted to elucidate the mechanism of DMTS formation during sake storage, but the mechanism underlying DMTS formation remains unclear. In this study, we determined the sake-preparation conditions that affect DMTS formation. We analyzed 76 sake samples immediately after filtration, which were donated by sake-producing companies. We measured the DMTS concentration in sake after 7 days of storage at 70°C (DMTS-pp) using gas chromatography/mass spectrometry. In the statistical analysis, DMTS-pp was set as the objective variable, whereas the preparation conditions and analytical results for sake were set as the explanatory variables. We used multiple linear regression (MLR) analysis with a stepwise method and partial least squares regression (PLSR) to analyze the data. The statistical analysis showed that the significant factors for DMTS-pp were the average temperature in the moromi mash (Temp ave), the total daily temperature in the moromi mash (Temp sum), the concentration of sulfur-containing amino acids in sake, and the Zn concentration in sake. These factors explained 63.4% of the variance in DMTS-pp according to the MLR analysis and 64.2% according to the PLSR analysis. Further MLR analysis showed that Temp ave in early stage and Temp sum in later stage were important factors for DMTS-pp. This result suggests that the rice dissolution caused by high Temp ave in early stage and yeast cell lysis caused by high Temp sum in later stage contribute to high DMTS-pp.

Fission yeast leucine-rich repeat protein Lrp1 is essential for cell morphogenesis as a component of the morphogenesis Orb6 network (MOR).

In eukaryotes, cell morphogenesis is regulated coordinately with the cell cycle. In fission yeast, the morphogenesis network MOR (morphogenesis Orb6 network) consists of 5 conserved proteins, Pmo25, Nak1, Mor2, Orb6, and Mob2, and is essential for cell polarity control and cell separation following cytokinesis. Here we show that the conserved leucine-rich repeat protein Lrp1 is required for cell morphogenesis as a newly recognized component of MOR. Lrp1 has 4 leucine-rich repeats in its N-terminus and is a homolog of the budding yeast Sog2, which is a component of the RAM network (regulation of Ace2 activity and cellular morphogenesis). Lrp1 was essential for both cell growth and cell morphogenesis as were the other MOR components. Lrp1 was localized to the SPBs (spindle pole bodies, the yeast equivalent of the animal centrosome) throughout the cell cycle and to the medial ring during cytokinesis. Lrp1 interacted with Nak1 and was important for Orb6 kinase activity. Thus Lrp1 proved to function upstream of Orb6 in cell morphogenesis.

Adenosine kinase-deficient mutant of Saccharomyces cerevisiae accumulates S-adenosylmethionine because of an enhanced methionine biosynthesis pathway.

To isolate an S-adenosylmethionine (SAM)-accumulating yeast strain and to develop a more efficient method of producing SAM, we screened methionine-resistant strains using the yeast deletion library of budding yeast and isolated 123 strains. The SAM content in 81 of the 123 strains was higher than that in the parental strain BY4742. We identified ADO1 encoding adenosine kinase as one of the factors participating in high SAM accumulation. The X∆ado1 strain that was constructed from the X2180-1A strain (MAT a, ATCC 26786) could accumulate approximately 30-fold (18 mg/g dry cell weight) more SAM than the X2180-1A strain in yeast extract peptone dextrose medium. Furthermore, we attempted to identify the molecular basis underlying the differences in SAM accumulation between X∆ado1 and X2180-1A strains. DNA microarray analysis revealed that the genes involved in the methionine biosynthesis pathway, phosphate metabolism, and hexose transport were mainly overexpressed in the X∆ado1 strain compared with the X2180-1A strain. We also determined the levels of various metabolites involved in the methionine biosynthesis pathway and found increased content of SAM, tetrahydrofolate (THF), inorganic phosphate, polyphosphoric acid, and S-adenosylhomocysteine in the X∆ado1 strain. In contrast, the content of 5-methyl-THF, homocysteine, glutathione, and adenosine was decreased. These results indicated that the ∆ado1 strain could accumulate SAM because of preferential activation of the methionine biosynthesis pathway.

Calcineurin ensures a link between the DNA replication checkpoint and microtubule-dependent polarized growth.

Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (+TIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signalling pathway linking +TIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The +TIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis.

Formation of spray-dried powder of S-adenosyl-L-methionine.

S-Adenosyl-L-methionine (SAM) is an essential metabolite in all living organisms. In clinical research, SAM has also been suggested as a chemotherapeutic agent in various diseases. The main problem of SAM is its instability at high temperatures, at neutral and alkaline pH, and in the presence of humidity. SAM retention in spray-dried powder was determined under various conditions of spray-drying. The highest SAM retention was obtained when maltodextrin (dextrose equivalent, DE, of 25) was used as the carrier solid with the SAM feed liquid at pH 4.0. The water content in the powder had a significant effect on the stability of SAM. SAM powder with lower water content exhibited higher stability.

The V260I mutation in fission yeast alpha-tubulin Atb2 affects microtubule dynamics and EB1-Mal3 localization and activates the Bub1 branch of the spindle checkpoint.

We have identified a novel temperature-sensitive mutant of fission yeast alpha-tubulin Atb2 (atb2-983) that contains a single amino acid substitution (V260I). Atb2-983 is incorporated into the microtubules, and their overall structures are not altered noticeably, but microtubule dynamics is compromised during interphase. atb2-983 displays a high rate of chromosome missegregation and is synthetically lethal with deletions in a subset of spindle checkpoint genes including bub1, bub3, and mph1, but not with mad1, mad2, and mad3. During early mitosis in this mutant, Bub1, but not Mad2, remains for a prolonged period in the kinetochores that are situated in proximity to one of the two SPBs (spindle pole bodies). High dosage mal3(+), encoding EB1 homologue, rescues atb2-983, suggesting that Mal3 function is compromised. Consistently, Mal3 localization and binding between Mal3 and Atb2-983 are impaired significantly, and a mal3 single mutant, such as atb2-983, displays prolonged Bub1 kinetochore localization. Furthermore in atb2-983 back-and-forth centromere oscillation during prometaphase is abolished. Intriguingly, this oscillation still occurs in the mal3 mutant, indicating that there is another defect independent of Mal3. These results show that microtubule dynamics is important for coordinated execution of mitotic events, in which Mal3 plays a vital role.