TY - JOUR
AU - Takagi, Hiroshi
AB - ABSTRACT In response to environmental stress, microorganisms adapt to drastic changes while exerting cellular functions by controlling gene expression, metabolic pathways, enzyme activities, and protein–protein interactions. Microbial cells that undergo a fermentation process are subjected to stresses, such as high temperature, freezing, drying, changes in pH and osmotic pressure, and organic solvents. Combinations of these stresses that continue over long terms often inhibit cells’ growth and lead to their death, markedly limiting the useful functions of microorganisms (eg their fermentation ability). Thus, high stress tolerance of cells is required to improve productivity and add value to fermented/brewed foods and biofuels. This review focuses on stress tolerance mechanisms, including l-proline/l-arginine metabolism, ubiquitin system, and transcription factors, and the functional development of the yeast Saccharomyces cerevisiae, which has been used not only in basic science as a model of higher eukaryotes but also in fermentation processes for making alcoholic beverages, food products, and bioethanol. Graphical Abstract Open in new tabDownload slide Stress tolerance mechanisms found in Saccharomyces cerevisiae can contribute to breeding of industrial yeast strains with improved fermentation productivity. Graphical Abstract Open in new tabDownload slide Stress tolerance mechanisms found in Saccharomyces cerevisiae can contribute to breeding of industrial yeast strains with improved fermentation productivity. yeast stress tolerance, proline/arginine metabolism, nitric oxide, ubiquitin system, transcription factor Abbreviations Abbreviations AcCoA: acetyl-CoA Arg: l-arginine Art: arrestin-related trafficking adaptor AVT: amino acid vacuolar transport AZC: l-azetidine-2-carboxylate CHOP: cis-4-hydroxy proline Cit: l-citrulline DARP: 2,5-diamino-6-(5-phospo-d-ribosylamino)-pyrimidin-4(3H)-one GK: γ-glutamyl kinase GM: genetically modified GSA: l-glutamate-γ-semialdehyde GSH: glutathione GSNO: S-nitrosoglutathione GSNOR: S-nitrosoglutathione reductase GTPCH2: GTP cyclohydrolase II HPLC/FLD: high-performance liquid chromatography with fluorescence detection MRC: mitochondrial respiratory chain NIR: nitrite reductase NO: nitric oxide NOD: NO dioxygenase NOS: NO synthase NR: nitrate reductase Orn: l-ornithine P5C: l-Δ1-pyrroline-5-carboxylate PCR: polymerase chain reaction Pro: l-proline ROS: reactive oxygen species RSNO: S-nitrosothiols SC: self-cloning α-syn: α-synuclein TCA: tricarboxylic acid Ub: ubiquitin Environmental stresses, such as nonoptimal growth temperatures, prolonged nutrient starvation, osmotic pressure and pH imbalances, and exposure to toxic molecules and free radicals, continuously challenge all livings cells (Mager and De Kruijff 1995). Microbial cells,  which are present in and on humans and other animals and in soil, water, and the air,  have developed a series of stress-responsive systems to withstand such adverse environmental conditions (Estruch 2000). These systems exist at the transcriptional, protein, and metabolic levels. For example, microbial cells perceive and transduce some external stimuli via signal transduction pathways, resulting in the global remodeling of gene expression, which itself is governed by the cells’ transcriptional activators and repressors. Stress applied to cells generally affects both transcription and translation activities, which results in an inhibition of de novo protein synthesis. Protein quality control and protein homeostasis are essential for the cellular responses to stress, since fluctuations in environmental parameters can damage proteins; examples of such damage are a destabilization of cellular structures, inhibition of the activities of enzymes, and chemical gradient instability, any of which will eventually result in disruption of the cells. In response to harsh stress, energy-producing and energy-consuming processes in cells are downregulated and enter a quiescent state. A dynamic shift in the central metabolic pathways that convert nutrients into energy and biomass often accompanies this state. The budding yeast Saccharomyces cerevisiae is a single-cell eukaryote that has been studied in basic science for decades. For example, the 2016 Nobel Prize in Physiology or Medicine was awarded to Yoshinori Ohsumi for his discoveries of the autophagy mechanisms in yeast cells (Długońska 2017). Yeast microbes have been also used in fermentation processes to make alcoholic beverages, bread, and fermented food products for hundreds of years. More recently, yeast has been used in the production of bioethanol. Our research group recently isolated yeast mutants that overproduce isoamyl acetate, which is known as the key ingredient providing the flavor of sake, which is made from fermented rice (Takagi et al. 2015; Abe et al. 2019). By brewing with these mutants, several kinds of awamori, which is a traditional distilled alcohol beverage in Okinawa Islands, have been recently commercialized. Yeast cells are subjected to a variety of environmental stresses during fermentation processes, and severe stress conditions can reduce their fermentation efficiency. The cellular tolerance to various types of stress is thus desired for their use in biotechnological applications. Freezing and other extreme temperatures, high ethanol concentrations, high osmotic pressure, and drying are some of the stresses to which yeast may be subjected during fermentation, and this stress can induce both the denaturation of proteins and the generation of reactive oxygen species (ROS) due to mitochondrial damage, inhibiting the yeast cells’ growth and/or causing their death. In fact, we observed a significant increase in intracellular ROS levels under a variety of stress conditions that mimic fermentation processes, indicating that yeast cells are commonly exposed to oxidative stresses during fermentation (Kitagaki and Takagi 2014). In the present review, I focus on my research group's findings regarding several stress tolerance mechanisms in yeast and the highly functional development of yeast. The physiological roles and metabolic regulations of the amino acids l-proline (Pro) and l-arginine (Arg) in stress tolerance are described, as is the mechanism underlying the repair or degradation of stress-induced abnormal proteins via the ubiquitin system. Stress-related transcription factors that can be manipulated for the improvement of stress tolerance are also presented. The mechanisms underlying stress tolerance via l-proline/l-arginine metabolism The metabolic regulations of l-proline in yeast The amino acid Pro serves as both a source of nitrogen and a stress protectant in S. cerevisiae (Takagi 2008). It is used by this yeast as a carbon source via the mitochondrial Pro metabolic pathway and the subsequent tricarboxylic acid (TCA) cycle, as our group recently demonstrated (Nishida, Watanabe and Takagi 2016a). The putative mitochondrial α-ketoglutarate-dependent dioxygenase Fmp12 inhibits the growth of yeast cells by avoiding the ATP production step in the TCA cycle, as suggested by the results of growth-phenotype analyses using gene disruption and overexpression (Nishida, Watanabe and Takagi 2016a). The metabolic regulation of Pro, including its biosynthesis, cellular localization, transport, and degradation, is thus of great interest. S. cerevisiae cells synthesize Pro mainly from l-glutamate (Glu) via 3 cytoplasmic enzymes: γ-glutamyl kinase (GK) (Pro1); γ-glutamyl phosphate reductase (Pro2); and l-Δ1-pyrroline-5-carboxylate (P5C) reductase (Pro3) (Figure 1). The activity of GK is subjected to feedback inhibition by Pro, indicating that GK is the rate-limiting enzyme that controls the intracellular level of Pro (Sekine et al. 2007). Pro is degraded generally in the mitochondria, but the import mechanism responsible for this is not known; the process involves mitochondrial Pro oxidase (Put1) converting Pro into P5C, which is then processed into Glu by P5C dehydrogenase (Put2). S. cerevisiae cells can also synthesize Pro from Arg via l-ornithine (Orn) catalyzed by Orn aminotransferase (Car2) in the cytoplasm (Kaino et al. 2012). Figure 1. Open in new tabDownload slide Metabolic pathways of l-proline (Pro) and l-arginine (Arg) in S. cerevisiae. Protein names: Pro1, γ-glutamyl kinase; Pro2, γ-glutamyl phosphate reductase; Pro3, Δ1-pyrroline-5-carboxylate (P5C) reductase; Put1, Pro oxidase; Put2, P5C dehydrogenase; Arg2, N-acetyl-glutamate synthase; Arg6, N-acetyl-glutamate kinase; Arg5, N-acetyl-glutamyl-5-phosphate reductase; Arg8, N-acetyl-ornithine aminotransferase; Arg7, N-acetyl-ornithine acetyltransferase; Arg11, Ornithine transporter; Arg3, ornithine carbamoyltransferase; Arg1, arginosuccinate synthetase; Arg4, arginosuccinate lyase; Car1, arginase; Car2, ornithine aminotransferase; Mpr1, N-acetyltransferase; and Tah18, flavoprotein (diflavin reductase family). Mainly, Pro and Arg are synthesized from l-glutamate. In response to high-temperature stress, nitric oxide (NO) is produced from the increased Arg through the Mpr1- and Tah18-dependent manner. Activity of Pro1 or Arg2, Arg6, and Arg5 is subject to feedback inhibition by Pro or Arg, respectively. Figure 1. Open in new tabDownload slide Metabolic pathways of l-proline (Pro) and l-arginine (Arg) in S. cerevisiae. Protein names: Pro1, γ-glutamyl kinase; Pro2, γ-glutamyl phosphate reductase; Pro3, Δ1-pyrroline-5-carboxylate (P5C) reductase; Put1, Pro oxidase; Put2, P5C dehydrogenase; Arg2, N-acetyl-glutamate synthase; Arg6, N-acetyl-glutamate kinase; Arg5, N-acetyl-glutamyl-5-phosphate reductase; Arg8, N-acetyl-ornithine aminotransferase; Arg7, N-acetyl-ornithine acetyltransferase; Arg11, Ornithine transporter; Arg3, ornithine carbamoyltransferase; Arg1, arginosuccinate synthetase; Arg4, arginosuccinate lyase; Car1, arginase; Car2, ornithine aminotransferase; Mpr1, N-acetyltransferase; and Tah18, flavoprotein (diflavin reductase family). Mainly, Pro and Arg are synthesized from l-glutamate. In response to high-temperature stress, nitric oxide (NO) is produced from the increased Arg through the Mpr1- and Tah18-dependent manner. Activity of Pro1 or Arg2, Arg6, and Arg5 is subject to feedback inhibition by Pro or Arg, respectively. We have reported the importance of the localization of Pro in vacuoles. An S. cerevisiae mutant with intracellular Pro accumulation cultured in a minimal medium also accumulated Pro in its vacuoles (Matsuura and Takagi 2005). Deletion of the PEP3 gene, which encodes a vacuolar membrane protein that is required for vacuolar biogenesis, conferred stress sensitivity to yeast cells, indicating that a specific vacuolar function is involved in stress-protective effects of Pro. Seven AVT genes (AVT1-7) in S. cerevisiae encode vacuolar amino acid transporters that are members of the amino acid vacuolar transport (AVT) family. In response to the application of exogenous Pro, these genes were significantly upregulated. We added Pro to S. cerevisiae cells grown in a minimal medium and then observed the effects of both the deletion and the overexpression of the AVT genes on the subcellular distribution of amino acids (Nishida et al. 2016b). The results indicated that Avt3 is the major protein involved in the export of Pro from the vacuole, and that the AVT genes induced by exogenous Pro are involved in the bidirectional transport of Pro across the vacuolar membrane. Pro is a predominant amino acid in grape must (the freshly crushed juice that contains the grapes’ skins, seeds, and stems), but Pro is not utilized effectively by S. cerevisiae in wine-making processes, resulting in a nitrogen deficiency during fermentation and the accumulation of Pro in the wine. We recently revealed that Arg inhibits the utilization of Pro by specifically inducing the endocytosis of the high-affinity Pro transporter Put4 (Nishimura, Tanikawa and Takagi 2020b). We recently revealed that Arg inhibits the utilization of Pro by specifically inducing the endocytosis of Put4, a high-affinity Pro transporter. A key regulator for Put4 endocytosis is an adaptor protein for the ubiquitin ligase Rsp5, ie Art3 (Nishimura, Tanahashi and Takagi 2020a). We discovered that deletion of the ART3 gene thoroughly canceled inhibition of Pro utilization by Arg. These findings may contribute to the development of strains of wine yeast that can efficiently assimilate the abundant Pro in grape must during the fermentation processes. The physiological functions of l-proline in yeast Several types of bacteria and plants accumulate Pro as an osmoprotectant (ie a compatible solute) in response to osmotic stress (Verbruggen and Hermans 2008). In contrast, yeast cells do not increase their level of intracellular Pro in response to various stress conditions including osmotic stress; rather, they respond by synthesizing glycerol or trehalose (Kaino and Takagi 2008). The functions of Pro that have been observed in vitro include a reduction of the melting temperature (Tm) of DNA in response to a destabilization of the double helix in salinity–stress tests, plus the stabilization of proteins and the cell membrane against elevated temperatures, freezing, and/or dehydrations (Takagi 2008). Pro also inhibits protein aggregation during its refolding or folding (Ignatova and Gierasch 2006), increases the solubility of sparingly soluble proteins, and scavenges ROS (Takagi 2008), particularly hydroxyl radical (Smirnoff and Cumbes 1989). In plants subjected to osmotic stress, the increase in Pro was shown to reduce the levels of free radicals (Hong et al. 2000). In cultured tobacco cells, treatment with Pro conferred tolerance to salinity and cadmium stress (Islam et al. 2009). The mechanisms underlying these functions in vivo are not established. In S. cerevisiae, the oxidation level in wild-type cells was clearly increased after ethanol exposure, suggesting the generation of ROS. Notably, in yeast cells at the stationary phase subjected to ethanol stress, the accumulation of Pro significantly reduced the level of ROS and increased the cell survival rate (Takagi, Taguchi and Kaino 2016). Pro and Arg are both known to exert significant cryoprotective activity that is nearly equal to those of glycerol and trehalose, which are major cryoprotectants for S. cerevisiae (Takagi, Iwamoto and Nakamori 1997). It has been speculated that Pro inhibits ice crystal formation and dehydration by forming strong hydrogen bonds with intracellular free water, probably due to the extremely high water solubility of Pro. We previously isolated or constructed S. cerevisiae strains that accumulated Pro, and showed that these strains enhanced tolerance to many types of stress treatments including ethanol, hydrogen peroxide, desiccation, and freeze-thawing (Takagi, Iwamoto and Nakamori 1997, 2000a, 2000b; Morita, Nakamori and Takagi 2001; Morita, Nakamori and Takagi 2003; Terao, Nakamori and Takagi 2003). Interestingly, the Asp154Asn and Ile150Thr variants of GK (the PRO1 gene product) were less sensitive to feedback inhibition, leading to the oversynthesis of Pro. The yeast cells that expressed these 2 variants could therefore accumulate Pro, and exhibited higher tolerance to the freeze-thawing stress (Figure 2a and b) (Sekine et al. 2007). The appropriate level of intracellular Pro might thus be important for cell protection against stress in yeast (Matsuura and Takagi 2005; Takagi, Taguchi and Kaino 2016). Figure 2. Open in new tabDownload slide Physiological functions of Pro and GK in S. cerevisiae. (a) Effect of Pro on the GK activity. The activities of various GKs were measured using the hydroxamate assay in the presence of the Pro concentrations indicated. The enzyme activities in the absence of Pro were defined as 100%. The values are the means of results from 3 independent experiments (Sekine et al. 2007). (b) Freezing stress tolerance of S. cerevisiae strains expressing GK. The cell viabilities of each strain were measured after freezing at −20 °C for the times indicated. The number of colonies before freezing was defined as 100%. The values are the means of results from 3 independent experiments (Sekine et al. 2007). (c) Proposed model for regulatory mechanism of ribophagy in yeast. First, nitrogen starvation induces cell death and ribophagy mediated by the Ubp3–Bre5 complex and GK. GK activity is required not only for Pro biosynthesis, but also for ribophagy. Finally, ribophagy suppresses cell death under nitrogen-starvation conditions (Tatehashi et al. 2016). Figure 2. Open in new tabDownload slide Physiological functions of Pro and GK in S. cerevisiae. (a) Effect of Pro on the GK activity. The activities of various GKs were measured using the hydroxamate assay in the presence of the Pro concentrations indicated. The enzyme activities in the absence of Pro were defined as 100%. The values are the means of results from 3 independent experiments (Sekine et al. 2007). (b) Freezing stress tolerance of S. cerevisiae strains expressing GK. The cell viabilities of each strain were measured after freezing at −20 °C for the times indicated. The number of colonies before freezing was defined as 100%. The values are the means of results from 3 independent experiments (Sekine et al. 2007). (c) Proposed model for regulatory mechanism of ribophagy in yeast. First, nitrogen starvation induces cell death and ribophagy mediated by the Ubp3–Bre5 complex and GK. GK activity is required not only for Pro biosynthesis, but also for ribophagy. Finally, ribophagy suppresses cell death under nitrogen-starvation conditions (Tatehashi et al. 2016). New physiological functions of Pro and GK were recently reported. We observed that intracellular Pro regulates the replicative lifespan of S. cerevisiae (Mukai et al. 2019). The deletion of PUT1 and the expression of PRO1I150T, which encodes the Ile150Thr variant of GK, resulted in the accumulation of Pro and extended the lifespan of yeast cells. Inversely, we found that the disruption of the Pro biosynthetic genes PRO1, PRO2, and CAR2 decreased the stationary Pro level and shortened the cellular lifespan. Pro metabolism may thus have a physiological role in maintaining the lifespan of yeast cells. In the nematode Caenorhabditis elegans, higher Pro levels increased longevity (Edwards et al. 2015). Impaired insulin/insulin-like growth factor (IGF-1) signaling was shown to extend the lifespan of C. elegans by promoting mitochondrial Pro catabolism, and Pro supplementation also extended the lifespan (Zarse et al. 2012). Although these observations indicated that Pro directly affects longevity in C. elegans, it should be noted that in yeast, changes in amino acid metabolism caused by the intracellular Pro might also be involved in longevity. A novel function of GK that is independent of the biosynthesis of Pro was suggested by the finding that the deletion of PRO1 leads to high sensitivity to various stresses, but the deletion of PRO2, which encodes γ-glutamyl phosphate reductase, does not. We demonstrated that PRO1 genetically interacts with UBP3, which encodes ubiquitin-specific protease and is required for the selective autophagy of ribosomes (ribophagy) (Figure 2c) (Tatehashi, Watanabe and Takagi 2016). Interestingly, GK activity is indispensable for ribophagy, which is important for cell survival during nitrogen starvation, while nonselective autophagy is not. The stress tolerance and fermentation ability of industrial yeasts with l-proline accumulation In the fermentation process used to produce Japanese sake, yeast cells are exposed to high ethanol concentrations that block the cell growth, viability, and fermentation (Kunkee and Bisson 1993). An appropriate Pro level in yeast cells can protect against ethanol stress, and the development of ethanol-tolerant sake yeasts could therefore reduce the time needed for fermentation. In addition, the taste of sake is derived from the amino acids that the yeast produces, and the creation of yeast strains with an added accumulation of “sweet” Pro could thus expand the range of sake flavors. A novel sake yeast strain was constructed by (i) replacing the wild-type PRO1 allele with the PRO1D154N allele encoding the Asp154Asn variant of GK and (ii) disrupting the PUT1 gene encoding Pro oxidase, which is required for the utilization of Pro (Takagi et al. 2005). As expected, this strain accumulated Pro and was more tolerant to ethanol stress compared to the control strain. Moreover, the sake brewed with the Pro-accumulating strain contained 5 times more Pro than sake brewed with the control strain, with no effect on the fermentation profiles. The Pro-rich sake had a distinctive sweet and light flavor (unpublished results). Recombinant yeasts have been investigated for commercial use. Since many consumers have indicated objections to genetically modified (GM) yeasts, the use of self-cloning (SC) yeast that do not contain foreign genes or DNA sequences other than yeast DNA may be more acceptable. We have constructed SC diploid sake yeast strains that accumulate Pro (Takagi et al. 2007), and we observed that compared to the parent strain, the Pro-accumulating strains produced greater amounts of Pro in sake (approx. 3 times more Pro) (Figure 3a) and approx. 30% more total amino acids. The ethanol production rate of the Pro-accumulating strains was somewhat faster than that of the parent strain (Figure 3b). These results suggest that intracellular Pro improves the fermentation rate, and that fermentation times of Pro-accumulating yeast strains might be shorter than those of wild-type control strains. Figure 3. Open in new tabDownload slide Fermentation abilities of Pro-accumulating sake and baker's yeast strains. (a, b) Laboratory-scale sake brewing was performed with a sake mash consisting of 160 g of steamed rice, 40 g of koji rice, and 260 mL of water added in 3 steps. Each strain was grown in SD medium at 30 °C for 2 days under static conditions and inoculated into the mash. Fermentation profiles were monitored by a loss of weight in conjunction with CO2 evolution. The sake mash was centrifuged and the supernatant was obtained as sake. General components of the sake, including Pro (a) and ethanol (b), were analyzed by the standard method established by the Japanese National Tax Administration Agency. The values are the means and standard deviation of results from 3 independent experiments (Takagi et al. 2007). (c) The doughs were prefermented for 120 min at 30 °C and then frozen for 9 days at −20 °C. The frozen dough was thawed for 30 min at 30 °C, and the remaining CO2 gas production was measured. The gassing power before freezing was defined as 100%. The values are the means and standard deviation of results from 3 independent experiments (Kaino et al. 2008). (d) Fermentation ability in sweet dough (30% sucrose per weight of flour) was monitored by CO2 gas production. The total amounts of CO2 production after 2 h were measured. The gassing power of wild-type strain (WT) was defined as 100%. The values are the means and standard deviation of results from 3 independent experiments (Sasano et al. 2012d). Figure 3. Open in new tabDownload slide Fermentation abilities of Pro-accumulating sake and baker's yeast strains. (a, b) Laboratory-scale sake brewing was performed with a sake mash consisting of 160 g of steamed rice, 40 g of koji rice, and 260 mL of water added in 3 steps. Each strain was grown in SD medium at 30 °C for 2 days under static conditions and inoculated into the mash. Fermentation profiles were monitored by a loss of weight in conjunction with CO2 evolution. The sake mash was centrifuged and the supernatant was obtained as sake. General components of the sake, including Pro (a) and ethanol (b), were analyzed by the standard method established by the Japanese National Tax Administration Agency. The values are the means and standard deviation of results from 3 independent experiments (Takagi et al. 2007). (c) The doughs were prefermented for 120 min at 30 °C and then frozen for 9 days at −20 °C. The frozen dough was thawed for 30 min at 30 °C, and the remaining CO2 gas production was measured. The gassing power before freezing was defined as 100%. The values are the means and standard deviation of results from 3 independent experiments (Kaino et al. 2008). (d) Fermentation ability in sweet dough (30% sucrose per weight of flour) was monitored by CO2 gas production. The total amounts of CO2 production after 2 h were measured. The gassing power of wild-type strain (WT) was defined as 100%. The values are the means and standard deviation of results from 3 independent experiments (Sasano et al. 2012d). We recently isolated yeast mutants that are resistant to the Pro analogue, l-azetidine-2-carboxylate (AZC), derived from a diploid sake yeast strain. Some of these mutants produced more Pro in the brewed sake, and one of the mutants carried a novel mutation in the PRO1 gene encoding the Gln79His variant of GK (Murakami et al. 2020). This mutation resulted in extreme desensitization to feedback inhibition by Pro, leading to an overproduction of Pro. The sake brewed with this mutant contained more Pro but less succinate than the sake brewed with the parent strain. A metabolome analysis indicated that the decrease in succinate was due to a lower level of 2-oxoglutarate, which is converted into Glu. This approach could be a practical method for breeding yeast strains to increase the diversity of sake flavors. Yeast strains for baking need high fermentation ability and durability in response to various baking methods. Baker's yeast cells are exposed to baking-associated stresses including high sucrose levels, freeze-thawing, and air-drying, all of which are believed to induce oxidative stress to the yeast cells (Attfield 1997; Shima and Takagi 2009). This oxidative stress may be attributable to the denaturation of proteins (eg antioxidant enzymes) and severe damage to the mitochondrial membrane and/or respiratory chain (Ando et al. 2007; Landolfo et al. 2008; Shima, Ando and Takagi 2008). Dried yeast is generally used in baking processes because it can be stored for longer periods and is less costly to transport compared to compressed yeast. Sweet (high-sugar) dough is up to 40% sucrose per weight of flour. The widely available frozen-dough technology can supply oven-fresh bakery products to consumers. Kaino et al. (2008) constructed SC diploid baker's yeast strains that accumulate Pro. They achieved this by replacing the wild-type PRO1 gene encoding GK with the PRO1D154N or PROI150T allele and disrupting the PUT1 gene (Figure 3c, and d). When the dough was prefermented before freezing and then kept frozen, the gassing power of wild-type cells was greatly decreased compared to the power observed before freezing. On the other hand, the cells that accumulated Pro showed ∼50% greater fermentation ability compared to the wild-type cells, indicating that baker's yeast with Pro accumulation has promise for the frozen-dough baking industry (Figure 3c). Compared to baker's yeast strains that accumulated only Pro or only trehalose, strains that simultaneously accumulated Pro and trehalose showed higher fermentation ability in frozen dough (Sasano et al. 2012a). Another study examined the results of inoculating baker's yeast cells into a liquid fermentation medium that contained a high level of sucrose. Under this stress condition, the level of ROS increased (Sasano et al. 2012d). This was also observed with wine yeast (Landolfo et al. 2007). Pro-accumulating SC diploid baker's yeast strains were constructed by Sasano et al. (2012d), and their findings demonstrated that Pro accumulation gives yeast cells greater tolerance to high-sucrose stress. When they evaluated baker's yeasts that are used in sweet dough, the Pro-accumulating strain showed an approx. 40% increase in gassing power compared to the wild-type strain (Figure 3d). Thus, Pro-accumulating baker's yeast strains are superior for the production of sweet bread. Dried yeast is exposed to air-drying stress during the preparation process. Air-drying stress is actually a combination of 2 stresses: high temperature and dehydration, which cause the accumulation of intracellular ROS (Franca, Panek and Eleutherio 2007). Air-drying also presents disadvantages that reduce fermentation ability, including mitochondrial malfunction, vacuolar acidification, and protein misfolding (Shima, Ando and Takagi 2008). Tolerance to air-drying stress is thus a necessary characteristic of the baker's yeast used in dried yeast preparations. We demonstrated that fermentation ability of baker's yeast after air-drying was significantly enhanced by the accumulation of Pro in the cells (Sasano et al. 2010). The methods used for breeding baker's yeasts are generally more acceptable to consumers since SC yeasts do not need to be treated the same as GM yeasts. Mutant yeast strains that are resistant to the Pro analogue AZC were isolated from diploid baker's yeast (Tsolmonbaatar et al. 2016), and under freezing or high-sucrose stress conditions some of these strains that accumulated Pro showed higher cell viability compared to the parent wild-type strain. It also appeared that most of the mutant Pro-accumulating strains had enhanced fermentation ability in frozen and sweet doughs. Two of those strains carried novel mutations in the PRO1 gene encoding the Pro247Ser or Glu415Lys variant of GK. Interestingly, these mutations produced resistance to AZC in the yeast cells as well as desensitization to the Pro feedback inhibition of GK, leading to an intracellular accumulation of Pro. Compared to the baker's yeast cells that expressed the wild-type PRO1 gene, baker's yeast cells expressing the PRO1P247S or PRO1E415K gene demonstrated higher tolerance to freezing stress. This approach could thus be effective for breeding Pro-accumulating baker's yeast strains that have higher tolerance to baking-associated stresses. Figure 4 shows the positions of amino acid residues in GK where mutations occur in the Pro-accumulating strains. Figure 4. Open in new tabDownload slide Features of GK variants. (a) Schematic representation of the S. cerevisiae GK. The positions of amino acids where mutations occur in the Pro-accumulating mutants are indicated by arrowheads. The numbers are residue numbers. The kinase and PUA domains are green and yellow, respectively. (b) Homology model of S. cerevisiae GK (Pro1, UniProtKB accession number P32264) was constructed by SWISS-MODEL using the structure of Escherichia coli GK bound with γ-glutamyl phosphate and oxoproline (39% sequence identity, PDB ID: 2J5V) as the template. Asp154, Ile150, and Gln79 (Sekine et al. 2007; Murakami et al. 2020) are shown in a stick model. (c) The structure of S. cerevisiae GK was predicted by homology modeling on I-TASSER based on the structure of E.coli GK (PDB ID: 2j5t) as the template. Asp154, Ile150, Pro247, and Glu415 (Sekine et al. 2007; Tsolmonbaatar et al. 2016) are shown in a stick model. Figure of GK structure was drawn using UCSF chimera. Some residues or the substrate CHOP are shown in a stick model or sphere model, respectively. Figure 4. Open in new tabDownload slide Features of GK variants. (a) Schematic representation of the S. cerevisiae GK. The positions of amino acids where mutations occur in the Pro-accumulating mutants are indicated by arrowheads. The numbers are residue numbers. The kinase and PUA domains are green and yellow, respectively. (b) Homology model of S. cerevisiae GK (Pro1, UniProtKB accession number P32264) was constructed by SWISS-MODEL using the structure of Escherichia coli GK bound with γ-glutamyl phosphate and oxoproline (39% sequence identity, PDB ID: 2J5V) as the template. Asp154, Ile150, and Gln79 (Sekine et al. 2007; Murakami et al. 2020) are shown in a stick model. (c) The structure of S. cerevisiae GK was predicted by homology modeling on I-TASSER based on the structure of E.coli GK (PDB ID: 2j5t) as the template. Asp154, Ile150, Pro247, and Glu415 (Sekine et al. 2007; Tsolmonbaatar et al. 2016) are shown in a stick model. Figure of GK structure was drawn using UCSF chimera. Some residues or the substrate CHOP are shown in a stick model or sphere model, respectively. The molecular functions of the novel N-acetyltransferase Mpr1 found in yeast When Arg was used as the sole source of nitrogen in yeast, the fermentation rate was increased more than when ammonium sulfate or Glu used as the sole source (Gutiérrez et al. 2012); the addition of Arg also increased the fermentation rate (Thomas and Ingledew 1992). These findings suggest that the synthesis of Arg promotes fermentation. In a study of the effects of amino acids on freezing stress applied to yeast cells, Arg and other charged amino acids (Glu and Lys) provided higher freeze tolerance (Takagi, Iwamoto and Nakamori 1997). The mechanisms underlying cryoprotective effect of Arg are not yet clear. Arg may serve as an ion coating on the membrane components’ and proteins’ surfaces that prevents denaturation by the NH2 group in the molecules. The effect of intracellular charged amino acids on freeze tolerance in dough was examined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast (Shima et al. 2003). During the frozen-dough baking process, one of the arginase mutants accumulated higher levels of Arg and/or Glu and showed increased leavening ability, suggesting that freeze tolerance is enhanced by the disruption of the CAR1 gene. During my investigations of Pro, I noticed the N-acetyltransferase Mpr1, which uses cyclic secondary amines (AZC and cis-4-hydroxy Pro) as substrates (Figure 5a). Among the many laboratory strains of Saccharomyces, the Σ1278b background strain has 2 copies of the MPR gene, MPR1 and MPR2 (sigma 1278b gene for proline-analogue resistance). A single base substitution exists at position 254, leading to Gly and Glu at position 85 in MPR1 and MPR2, respectively; nevertheless, the gene products (Mpr1 and Mpr2) play similar roles in resistance to AZC (Takagi, Iwamoto and Nakamori 1997, 2000a, 2000b). The Mpr1 protein was revealed as a novel N-acetyltransferase that detoxifies AZC through its N-acetylation in the S. cerevisiae Σ1278b strain (Shichiri et al. 2001). The genomes of various yeasts and fungi have been shown to contain genes that are homologous to MPR1, and AZC acetyltransferase activity has been observed in many types of yeast. Mpr1 homologues thus appear to be widely conserved in yeasts and fungi (Kimura, Nakamori and Takagi 2002; Wada et al. 2008). Interestingly, our research group has demonstrated the involvement of Mpr1 in a novel Arg biosynthesis pathway, and we observed that Mpr1 may acetylate P5C or its tautomer l-glutamate-γ-semialdehyde (GSA) to provide N-acetyl-GSA, an intermediate of the Arg biosynthesis pathway (Nishimura et al. 2010) (Figure 5b), thereby promoting the presence of N-acetyl-GSA in yeast. These findings suggested that an Mpr1-mediated enhancement of Arg synthesis could enable the construction of new yeast strains with better fermentation rates. Figure 5. Open in new tabDownload slide Molecular profiles of Mpr1 found in S. cerevisiae. (a) Proposed scheme for the AZC acetyltransferase reaction by Mpr1. cis-4-Hydroxy Pro (CHOP) is also acetylated in the same manner. (b) Growth curves of yeast strains during cultivation in SD medium at 30 °C. Mpr1 restored growth in arg2-deficient cells, even in the absence of Arg, but could not compensate the arg8-disruption blocking the downstream pathway of N-acetyl glutamate (Nishimura et al. 2010). (c) The structure of Mpr1-CHOP (light blue). It is superimposed with that of an aminoglycoside 6′-N-acetyltransferase from Enterococcus faecium (PDB ID code 1B87) (salmon pink). AcCoA in the structure 1B87 and CHOP in the structure of Mpr1-CHOP are shown as a sphere model (Nasuno et al. 2013). (d) Schematic illustration of CHOP binding site on Mpr1. CHOP is shown by black color, and red letters indicate the carbon atom numbering of CHOP. The red and blue residues interact with the substrates through their side chain and backbone, respectively. Black dotted lines show the possible van der Waals interaction and hydrogen bonds. CHOP binds to the side chain of Asn135 and the backbone amide N-H group of Asn172 and Leu173 through its carboxyl group, and to a water molecule bound to Phe138 through its amine group. CHOP also forms van der Waals contact with the phenolic side chain of Tyr75 through its Cγ atom (Nasuno et al. 2013). Figure 5. Open in new tabDownload slide Molecular profiles of Mpr1 found in S. cerevisiae. (a) Proposed scheme for the AZC acetyltransferase reaction by Mpr1. cis-4-Hydroxy Pro (CHOP) is also acetylated in the same manner. (b) Growth curves of yeast strains during cultivation in SD medium at 30 °C. Mpr1 restored growth in arg2-deficient cells, even in the absence of Arg, but could not compensate the arg8-disruption blocking the downstream pathway of N-acetyl glutamate (Nishimura et al. 2010). (c) The structure of Mpr1-CHOP (light blue). It is superimposed with that of an aminoglycoside 6′-N-acetyltransferase from Enterococcus faecium (PDB ID code 1B87) (salmon pink). AcCoA in the structure 1B87 and CHOP in the structure of Mpr1-CHOP are shown as a sphere model (Nasuno et al. 2013). (d) Schematic illustration of CHOP binding site on Mpr1. CHOP is shown by black color, and red letters indicate the carbon atom numbering of CHOP. The red and blue residues interact with the substrates through their side chain and backbone, respectively. Black dotted lines show the possible van der Waals interaction and hydrogen bonds. CHOP binds to the side chain of Asn135 and the backbone amide N-H group of Asn172 and Leu173 through its carboxyl group, and to a water molecule bound to Phe138 through its amine group. CHOP also forms van der Waals contact with the phenolic side chain of Tyr75 through its Cγ atom (Nasuno et al. 2013). The results of our crystallographic analysis revealed that the overall structure of Mpr1 is based on folding that is typical among the proteins in the Gcn5-related N-acetyltransferase superfamily (Figure 5c) (Nasuno et al. 2013). Mpr1 is folded into an α/β structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA (AcCoA) binding site is located near the backbone amide of Phe138 and the side chain of Asn178 (Figure 5d). An alanine substitution of Asn178, which can interact with the sulfur of AcCoA, markedly reduced the apparent kcat value. In addition, the replacement of Asn135 led to a markedly increased apparent Km value. Together these results indicated that Asn178 plays an important role in catalysis, and Asn135 has an important role in substrate recognition (Nasuno et al. 2013). Structure-based molecular design enabled the creation of the 2 stable variants (ie Asn203Lys-Mpr1 and Asn203Arg-Mpr1) each of which exhibited longer half-life activity compared to the wild-type Mpr1 (Nasuno et al. 2016). Polymerase chain reaction (PCR) random mutagenesis was used to isolate the Phe65Leu variant, which led to a further stabilization of Mpr1 (Iinoya et al. 2009). In growth assays, the overexpression of stable Mpr1 variants in yeast cells appeared to increase the synthesis of Arg. The construction of new yeast strains with both higher Arg synthetic ability and improved fermentation ability can make use of these findings. Mpr1 was reported to protect yeast cells against various oxidative stress conditions by reducing their intracellular ROS levels (Nomura and Takagi 2004; Du and Takagi 2005, 2007). High temperatures cause oxidative stress by generating ROS in the mitochondria (Moraitis and Curran 2007), prompting strong transcriptions of MPR1 and PUT1 and leading to an increased intracellular level of Arg (Nishimura et al. 2010). The increased conversion of Pro into Arg endowed yeast cells with oxidative stress tolerance, indicating that an unknown antioxidative mechanism is involved in the stress-induced synthesis of Arg requiring Mpr1 and Put1 (Nishimura et al. 2010). The stress tolerance and fermentation ability of industrial yeasts with Mpr1 expression Interestingly, among the many yeast strains used in industry, the Japanese baker's yeast strains have 1 copy of the MPR2 gene on chromosome X (Sasano et al. 2010). For an examination of the role of MPR2 in baker's yeast, we tested cell viability of diploid industrial baker's yeast strains and intracellular ROS level after exposing the strains to air-drying stress. The results revealed that the MPR2 in baker's yeast is involved in the tolerance to air-drying stress by reducing the intracellular ROS levels (Figure 6a) (Sasano et al. 2010). We also observed that compared to the wild-type Mpr1, the fermentation ability of bread dough after its exposure to air-drying stress was increased by the expression of the Lys63Arg and Phe65Leu variants with enhanced enzymatic functions (Figure 6a). The antioxidant enzyme Mpr1 thus has potential for the breeding of novel yeast strains that are tolerant to air-drying stress. Figure 6. Open in new tabDownload slide Effects of the wild-type and variant of Mpr1 on baker's and sake yeast strains. (a) Fermentation ability of baker's yeast strains after air-drying. Compressed yeast was treated with air-drying stress for 4 h at 37 °C. The dough containing the stress-treated yeasts was fermented for 3 h, and the remaining CO2 gas production was measured. The amount of CO2 production of the wild-type strain (WT) after air-drying stress treatment was defined as 100%. The values are the means and standard deviation of results from 3 independent experiments (Sasano et al. 2010). (b) Total CO2 emission (L) of sake yeast strain harboring the empty vector (EV), the wild-type Mpr1 (WT), and the Asn203Lys variant Mpr1 (N203K) in SC20-ura medium at 25 °C. The values are the means and standard deviation of results from 4 independent experiments. Statistically significant differences between N203K and WT or EV were determined by Student's t-test (*P < .05) (Ohashi et al. 2019). Figure 6. Open in new tabDownload slide Effects of the wild-type and variant of Mpr1 on baker's and sake yeast strains. (a) Fermentation ability of baker's yeast strains after air-drying. Compressed yeast was treated with air-drying stress for 4 h at 37 °C. The dough containing the stress-treated yeasts was fermented for 3 h, and the remaining CO2 gas production was measured. The amount of CO2 production of the wild-type strain (WT) after air-drying stress treatment was defined as 100%. The values are the means and standard deviation of results from 3 independent experiments (Sasano et al. 2010). (b) Total CO2 emission (L) of sake yeast strain harboring the empty vector (EV), the wild-type Mpr1 (WT), and the Asn203Lys variant Mpr1 (N203K) in SC20-ura medium at 25 °C. The values are the means and standard deviation of results from 4 independent experiments. Statistically significant differences between N203K and WT or EV were determined by Student's t-test (*P < .05) (Ohashi et al. 2019). We recently investigated the effects of Asn203Lys (a stable variant of Mpr1) on the ethanol fermentation of a sake yeast strain that lacks the MPR1 gene (Ohashi et al. 2019). The fermentation performance of this variant was improved compared to the wild-type Mpr1 when the variant was expressed in the diploid Japanese sake strain (Figure 6b). In laboratory-scale brewing, a sake strain that expresses the Asn203Lys variant produced more ethanol than the wild-type Mpr1. The Asn203Lys variant also affected the contents of flavor compounds and organic acids. This stable Mpr1 variant could therefore contributes to the construction of new industrial yeast strains with improved fermentation ability and diversity of taste and flavor. The physiological roles of nitric oxide in yeast The cellular signaling molecule nitric oxide (NO) is widely conserved among organisms, including microorganisms and higher eukaryotes. As a free radical gaseous molecule, NO participates in many biological processes in mammals such as protection against pathogens and the regulation of blood pressure (Astuti, Nasuno and Takagi 2018). NO has dual, concentration-dependent functions in both unicellular organisms and plants, and in bacteria, a low nanomolar cellular level of NO contributes to the self-defense against oxidative stress (Gardner et al. 2000). The formation of the biofilm is regulated by NO, thus promoting the formation of the bacterial colony (Allain et al. 2011). NO also has a role in the synthesis of toxins, and at higher levels NO can be toxic and induce cell death (Poole 2005). In yeasts, a low NO level mediates resistance to oxidative and temperature stress (Nishimura, Kawahara and Takagi 2013; Nasuno et al. 2014; Liu et al2015). At high micromolar levels, NO can enhance oxidative and nitrosative stress (Almeida et al. 2007; Tillmann, Gow and Brown 2011). A low level of NO is also important for the resistance to oxidative stress in plants (Considine, Sandalio and Foyer 2015). Based on its high diffusibility and reactivity, NO exerts opposing effects in cells, “acting as a double-edged sword”; for example, many physiological aspects of cardiovascular protection involve NO by activating a cGMP-mediated signal transduction pathway, and NO also enhances the tolerance of mammals and plants to oxidative stress by amplifying the cellular antioxidative activity (Petrovic et al. 2008; Martin et al. 2009). However, at higher NO levels or in the presence of superoxide, NO can have cytotoxic effects, potentially through the formation of the oxidant peroxynitrite, which induces cell damage via lipid peroxidation and protein inactivation by oxidation and nitration, leading to cardiovascular dysfunction. NO can react with other radical molecules, and this reaction generates more reactive secondary products, including reactive nitrogen species (RNS). The RNS can react with proteins that have free sulfhydryl groups at cysteine residues, and they react with tyrosine residues or with metals and heme to perform nitrosylation and nitration processes (Ridnour et al. 2004). Nitrosylation influences the activities of proteins, protein–protein interactions, and the localization of proteins (Benhar, Forrester and Stamler 2009). Cytoprotective actions exerted by NO have been reported in yeast cells subjected to a variety of environmental stress conditions, particularly oxidative stress. This cytoprotection is achieved by NO's regulation of certain transcriptional factors, including the S. cerevisiae Mac1 (Nasuno et al. 2014), the fission yeast Schizosaccharomyces pombe Pap1 (Kang et al. 2011), Sty1 (Astuti, Watanabe and Takagi 2016), and Rst2 (Kato, Zhou and Ma 2013), which modulate different mechanisms. For example, the activation of Mac1 by NO that was produced under a high-temperature-stress condition was shown to be important for the activation of the copper-dependent superoxide dismutase Sod1, which is a crucial antioxidative enzyme (Nasuno et al. 2014) (Figure 7a). In S. pombe, NO is localized in the mitochondria at the stationary phase, suggesting that 2 distinct types of NO signaling exist. For mitochondria, pretreatment with an NO donor rescued the cell growth by repressing the generation of ROS under oxidative stress. The results of a DNA microarray analysis of S. pombe demonstrated that exogenous NO contributed to the yeast's tolerance to H2O2 (hydrogen peroxide) by (i) inhibiting the conversions of Fe3+ to Fe2+, (ii) upregulating the H2O2-detoxifying enzymes, and (iii) downregulating the mitochondrial respiratory chain (MRC) genes. These findings indicated that NO plays a pivotal role in the negative feedback system to regulate ROS levels in S. pombe (Figure 7b) (Astuti, Watanabe and Takagi 2016). Figure 7. Open in new tabDownload slide Proposed models for NO-mediated oxidative stress responses in yeast. (a) When S. cerevisiae cells are exposed to high-temperature stress, NO is produced from Arg by an NOS-like activity. NO-mediated Mac1 is activated by post-translational modification, then upregulates the expression of CTR1, which encodes a copper transporter. Protein names: Fre1, cupric reductase; Ctr1, copper ion transporter; Sod1, Cu, Zn-superoxide dismutase; Ccs1, copper chaperone; Mac1, transcription factor responsible for copper metabolism (Nasuno et al. 2014). (b) In S. pombe, NO is mainly generated by the activities of an unidentified NOS and the MRC complex III during log phase growth and is detoxified by Yhb1 (NOD) and Fmd2 (GSNOR) in stationary phase. NO triggers at least 3 antioxidant mechanisms: (i) inhibition of the conversion of Fe3+ into Fe2+ (ii) upregulation of the genes encoding H2O2-detoxifying enzymes, and (iii) downregulation of the MRC genes (Astuti et al. 2016). Figure 7. Open in new tabDownload slide Proposed models for NO-mediated oxidative stress responses in yeast. (a) When S. cerevisiae cells are exposed to high-temperature stress, NO is produced from Arg by an NOS-like activity. NO-mediated Mac1 is activated by post-translational modification, then upregulates the expression of CTR1, which encodes a copper transporter. Protein names: Fre1, cupric reductase; Ctr1, copper ion transporter; Sod1, Cu, Zn-superoxide dismutase; Ccs1, copper chaperone; Mac1, transcription factor responsible for copper metabolism (Nasuno et al. 2014). (b) In S. pombe, NO is mainly generated by the activities of an unidentified NOS and the MRC complex III during log phase growth and is detoxified by Yhb1 (NOD) and Fmd2 (GSNOR) in stationary phase. NO triggers at least 3 antioxidant mechanisms: (i) inhibition of the conversion of Fe3+ into Fe2+ (ii) upregulation of the genes encoding H2O2-detoxifying enzymes, and (iii) downregulation of the MRC genes (Astuti et al. 2016). In S. cerevisiae, NO has a pivotal role in the tolerance of cells to high temperatures, heat shock, and hydrostatic pressure imbalance (Domitrovic et al. 2003). Our research has demonstrated that an increased NO level conferred tolerance to high-temperature stress (Nishimura et al. 2013; Nasuno et al. 2014). The activity of NOS is repressed by the loss of Cka2, which further decreases the cell viability under H2O2-induced apoptosis or high-temperature stress (Liu et al. 2015). Calorie restriction in S. cerevisiae induced a mitochondria-dependent synthesis of NO, leading to a metabolic shift that improved the cellular metabolism and the cellular response to an age-related accumulation of oxidative stress (Li et al. 2011). Those findings indicated that NO may help combat the effects of aging and age-related diseases. In addition, a comprehensive study using a yeast model revealed the pathology of Batten disease, a fatal disease of the nervous system that is the result of reduced NO production due to the loss of Cln3, a protein that is important for the maintenance of the intracellular Arg level and pH homeostasis (Osório et al. 2007). The synthetic regulations of nitric oxide in yeast Living organisms use 2 major mechanisms for NO production: (i) the Arg pathway catalyzed by NO synthases (NOSs) and (ii) the nitrite pathway, which provides a simple way to produce NO through the electron reduction of nitrate catalyzed by nitrate reductase (NR) and nitrite reductase (NIR). The NO in mammalian cells is endogenously synthesized from Arg by the enzymatic reaction of NOSs. Arg is oxidized to form l-citrulline (Cit) and NO, and NADPH and oxygen are involved in the reaction. Bacteria can also generate Arg-dependent NO through NOS systems. NOS-like proteins have been identified in many prokaryotes based on their genome sequences (Chen and Rosazza 1995; Adak, Aulak and Stuehr 2002; Hong et al. 2003; Kers et al. 2004; Gusarov et al. 2008; Agapie et al. 2009). Bacterial NOSs (bNOSs) are comprised of only an oxygenase domain, unlike mammalian NOSs (Adak, Aulak and Stuehr 2002; Gusarov et al. 2008; Bird et al. 2009). It is believed that evolutionarily, eukaryotic NOS originated earlier than bNOS, and that bNOS evolved to use a dedicated reductase domain (Gusarov et al. 2008). However, the bNOS of the soil-dwelling Gram-negative bacterium Sorangium cellulosum have both an oxygenase domain and a reductase domain, providing a new indication that the NOS present in both eukaryotes and prokaryotes evolved from a common ancestor (Agapie et al. 2009). The generation of NO via NOS-like activity in S. cerevisiae and S. pombe has been observed in both in vitro and in vivo analyses (Almeida et al. 2007; Kig and Temizkan 2009; Yoshikawa et al. 2016), but the corresponding NOS-encoding gene has not been identified. Our laboratory revealed the generation of NO in S. cerevisiae via an Arg-dependent mechanism that is mediated by the flavoprotein Tah18 (Nishimura, Kawahara and Takagi 2013) (Figure 1). We observed that in laboratory and baker's yeast strains, this particular Tah18-dependent NO generation was associated with the activities of MPR1 and PUT1 in the Pro-Arg metabolic pathway. This association is based on the following: the induction of these 2 genes led to a significant increase in the level of Arg (ie the NOS substrate), especially in response to environmental stressors such as air-drying, high temperatures, and freeze-thawing (Sasano et al. 2012c; Nishimura, Kawahara and Takagi 2013). Despite the finding that TAH18 expression is required for NOS-like activity in S. cerevisiae, the details of Tah18-associated NOS activity are not yet clear; this is because although Tah18 is homologous to the reductase domain, the Tah18 protein does not contain an intact oxygenase domain of NOS. Tah18 transfers electrons from NADPH to the Fe-S clusters of the Dre2 protein via flavin molecules (FAD and FMD), leading to the biogenesis of Fe-S cluster proteins (Netz et al. 2010). Our biochemical analyses recently showed that in response to oxidative stress Tah18 dissociates from Dre2, accompanied by an increase in the intracellular NO level. We also observed that under oxidative stress conditions the enhancement of the Tah18–Dre2 interaction suppressed the production of NO, indicating that the interaction of Tah18 with Dre2 inhibits the Tah18-dependent NOS-like activity (Yoshikawa et al. 2016). These findings suggest that (i) Tah18 may acts as a reductase domain to transfer electrons from NADPH to an unidentified oxygenase protein, which oxidizes Arg to Cit and NO, and (ii) Dre2 inhibits the Tah18-dependent NOS-like activity by removing electrons derived exclusively from NADPH. The Tah18-Dre2 complex might therefore function as a molecular switch to control NO production in response to environmental oxidative conditions (Figure 8) (Astuti, Watanabe and Takagi 2016; Astuti, Nasuno and Takagi 2018). Figure 8. Open in new tabDownload slide Dual effects of NO in S. cerevisiae. Like mammalian cells, Tah18-dependent NO production exhibits 2 opposed effects in yeast cells. For example, appropriate NO level confers tolerance to high temperature to cells (Nasuno et al. 2014). In contrast, under severe stress conditions, such as high levels of H2O2, excess NO induces cell death (Yoshikawa et al. 2016). Figure 8. Open in new tabDownload slide Dual effects of NO in S. cerevisiae. Like mammalian cells, Tah18-dependent NO production exhibits 2 opposed effects in yeast cells. For example, appropriate NO level confers tolerance to high temperature to cells (Nasuno et al. 2014). In contrast, under severe stress conditions, such as high levels of H2O2, excess NO induces cell death (Yoshikawa et al. 2016). Physiological functions and metabolism of NO in yeast remain to be clarified. A new method for obtaining more precise measurements of the NO content in S. cerevisiae cells with the detection limit of 6 n m was recently developed in our laboratory. The measurements are achieved by first treating the cells with an NO-specific fluorescence probe, and the performing high-performance liquid chromatography with fluorescence detection (HPLC/FLD) (Nasuno et al. 2020). With this method we successfully quantified the NO content inside yeast cells that were treated with an NO donor. Other results of this HPLC/FLD analysis indicated that the fluorescence that is induced under some stress conditions (eg heat-shock, ethanol, and vanillin treatment) was not derived from NO. The HPLC/FLD method measures the intracellular NO concentration with higher accuracy than has been available in the past. We also developed an analytical method to identify the nitrogen source for NO generation; this method uses liquid chromatography with tandem mass spectrometry and stable isotope labeling (Nasuno, Yoshikawa and Takagi 2021). We detected a 15N-labeled NO-containing compound generated by a 15N-labeled substrate nitrite both in vitro and in vivo by using this new method. The detoxification systems of nitric oxide in yeast Nitrosative stress is induced by the accumulation of NO derivatives, especially RNS (Ridnour et al. 2004). Several RNS molecules, such as peroxynitrite, the higher oxides of nitrogen, S-nitrosothiols (RSNO), and dinitrosyl iron complexes, are reported to have roles in cellular systems (Heck 2001). The mechanisms of intracellular NO detoxification are important for ensuring that NO can exert its cytoprotective actions rather than cytotoxic effects on cells. The maintenance of intracellular NO homeostasis involves S-nitrosoglutathione reductase (GSNOR) and NO dioxygenase (NOD) (Gardner 2005). S-nitrosoglutathione (GSNO) is often used as a marker of the intracellular level of RSNO, because glutathione (GSH, the substrate for GSNO synthesis) is the most abundant thiol-containing compound in eukaryotic cells. NOD (or flavohemoglobin) mediates deoxygenation by using the reductive power of NAD(P)H to covalently bond the 2 oxygen atoms in O2 with NO, yielding NO3− (Gardner 2005). Our latest study of S. pombe cells demonstrated that as NO-detoxification enzymes, the putative NO dioxygenase SPAC869.02c (named Yhb1) and the GSNO Fmd2 cooperatively reduced intracellular levels of NO (Astuti, Watanabe and Takagi 2016). Yhb1 is likely NOS-dependent, whereas Fmd2 is NOS-independent and may be associated with a mitochondrial-dependent production of NO. These two NO-detoxification enzymes are conserved among eukaryotes from yeast to mammals, indicating that NO is likely to be involved in primary physiological activities that are conserved throughout evolution. We recently identified a novel nitrosative stress tolerance gene in S. cerevisiae, RIB1, which encodes GTP cyclohydrolase II (GTPCH2) (Anam, Nasuno and Takagi 2020). The first step in riboflavin biosynthesis is catalyzed by GTPCH2. The GTPCH2 enzymatic activity of Rib1 is essential for the nitrosative stress tolerance that is RIB1-dependent, but riboflavin itself is not required for this tolerance. We also observed that the reaction mixture of recombinant purified Rib1 quenched NO or its derivatives, whereas formate or pyrophosphate (byproducts of the Rib1 reaction) did not. These findings suggested that the reaction product of Rib1, ie 2,5-diamino-6-(5-phospho-d-ribosylamino)-pyrimidin-4(3H)-one (DARP), scavenges NO or its derivatives. Our analyses demonstrated that 2,4,5-triamino-1H-pyrimidin-6-one, which is identical to a pyrimidine moiety of DARP, also scavenged NO or its derivatives, suggesting that DARP reacts with the dinitrogen trioxide (N2O3) generated via its pyrimidine moiety. Since there has been no report that DARP or another riboflavin metabolism intermediate scavenges RNS or attenuates nitrosative stress, we identified RIB1/DARP for the first time as a novel gene/metabolite that functions in the induction of the nitrosative stress tolerance mechanism in S. cerevisiae. Biotechnological applications of nitric oxide in yeast NO signaling is known to be involved in the pathology of several natural processes (eg aging) and degenerative diseases (eg neurodegenerative syndromes, cancer) (Khurana and Lindquist 2010). Investigations of yeast may help reveal the underlying mechanisms of these NO-related diseases, as a model of degenerative diseases, mitochondria-related diseases (Lasserre et al. 2015), and aging phenomena (Li et al. 2011). The findings regarding the involvement of NO in the stress response of industrially beneficial yeasts suggests that yeast cells could be modified by NO-targeted engineering at both the genetic and physiological levels. Modifications of the synthesis of NO designed to enhance the fermentation ability of baker's yeast have been described. For example, Sasano et al. (2012c) engineered a SC diploid baker's yeast strain with enhanced Pro and NO synthesis that also shows an increased intracellular NO level in response to air-drying stress; the strain better tolerates air-drying, oxidative, and freeze-thawing, which may be due to the strain's reduced intracellular ROS level. Compared to the wild-type strain, the new strain retained higher leavening activity in bread dough after air-drying and freeze-thawing. These characteristics suggest that (i) NO is synthesized in baker's yeast in response to oxidative stresses, and (ii) increased NO is important in baking-associated stress tolerance. The protein quality-control mechanism used by the ubiquitin system under stress conditions One of the fundamental cellular processes for regulating cellular activities at the post-translational level is protein ubiquitination, achieved through protein modification by the small regulatory protein ubiquitin (Ub), a highly conserved protein that contains 76 amino acids. Ubiquitination has roles in a variety of cellular processes such as regulation of the cell cycle and apoptosis, protein degradation and trafficking, and an array of signal transductions (Mukhopadhyay and Riezman 2007). The ubiquitination process occurs via a multistep cascade. As the first step, Ub is activated by the E1-activating enzyme in an ATP-dependent manner. The formation of a thioester bond between the C-terminal glycine residue of Ub and a cysteine in the active center of E1 then takes place. The activated Ub is transferred to a cysteine residue located at the active center of the E2-conjugating enzyme. The next step is the prompt transfer of the active Ub to the target protein with the aid of the E3 Ub ligase. As the process continues, the ligation of Ub with the target protein occurs via an isopeptide bond formed between the C-terminal glycine residue of Ub and the lysine residues of the target protein (or another Ub, if a poly-Ub chain is formed). Ub ligases can be categorized into 2 types: the RING (really interesting new genes)-finger type (Jackson et al. 2000) and the HECT (homologous to E6-AP carboxyl terminus) type (Huibregtse et al. 1995). The many HECT-type Ub ligases in S. cerevisiae include Hul4, Hul5, Ufd4, Tom1, and Rsp5 (Wang, Yang and Huibregtse 1999). The sole essential HECT-type Ub ligase in the Nedd4 family found in S. cerevisiae is Rsp5. The ubiquitin ligase Rsp5 Rsp5 (Reverses Spt− phenotype protein 5) is originally isolated as a mutated gene that suppresses SPT3 mutations that negatively affect the gene expression (Winston 1993). Rsp5 is the orthologue of the human Nedd4 Ub ligase in S. cerevisiae, and it is thus speculated to play important roles in signal transduction (Dunn and Hicke 2001), the quality control of plasma membrane proteins (Shiga et al. 2014), and intracellular trafficking (Jarmoszewicz et al. 2012). Rsp5 is composed of an N-terminal Ca2+-dependent phospholipid membrane binding (C2) domain, 3 substrate recognition (WW) domains (commonly referred to 2 conserved tryptophan residues in the domains), and the C-terminal catalytic (HECT) domain (Rotin and Kumar 2009). The C2 domain of Rsp5, comprised of ∼130 amino acids, is normally regulated by the Ca2+ ions. This domain is important for the ability of Rsp5 to electrostatically interact with either the endosomal membrane or plasma membrane (Cho 2001). Each of the WW domains of Rsp5 consists of ∼40 amino acid residues, as Sudol (1996) initially described. The WW domains interact with substrates or adaptor proteins by recognizing proline-rich sequences (PY motifs; Pro-Pro-X-Tyr) within these proteins. The three WW domains in Rsp5 are WW1, WW2, and WW3. The HECT domain is composed of ∼350 amino acid residues and accounts for the ubiquitination activity of Rsp5. The important residue in this catalytic domain is Cys777, which is the location for thioester formation with ubiquitin. The mutation of Cys777 to Ala thus abolishes the overall ubiquitination activity of Rsp5 (Huibregtse et al. 1995). The mutation of Leu733 to Ser, known as rsp5-1 (a temperature-sensitive allele) was reported to cause a defect in the Ub-thioester formation and decrease the stability of Rsp5 at restrictive temperatures (Wang, Yang and Huibregtse 1999). Rsp5 must interact with the substrate through an interaction between its WW domains and the PY motifs located in the substrate, but most of the substrates for Rsp5 do not contain PY motifs (Gupta et al. 2007). Thus, Rsp5 may interact with a substrate via non-PY motifs or with the assistance of adaptors that contain a PY-motif, eg the arrestin-related trafficking adaptor (Art) proteins (Léon and Haguenauer-Tsapis 2009). Yeasts possess 14 Art proteins (Art1-10, Bul1-3, and Spo23), all of which contain the PY motifs recognized by Rsp5. This arrangement functions to direct nonfunctional or misfolded proteins out of the plasma membrane and prevent their aggregation, thereby maintaining the plasma membrane's integrity under proteotoxic stress, such as heat shock (Zhao et al. 2013). Repair and degradation mechanisms of stress-induced abnormal proteins mediated by Rsp5 Cells have a quality-control mechanism for proteins that are damaged by stress; that is, a repair and degradation mechanism. To investigate cellular functions under various stress conditions during the fermentation processes, our research group isolated a laboratory AZC-hypersensitive mutant yeast strain that is sensitive to various stresses (Hoshikawa et al. 2003). This mutant carries an allele of RSP5, leading to a single-amino acid change within the WW3 domain (Ala401Glu). The addition of NH4+ to yeast cells growing on induced rapid ubiquitination, endocytosis and the vacuolar degradation of the plasma membrane protein (the general amino acid permease Gap1). The Gap1 in the rsp5A401E mutant remained stable and active on the plasma membrane (and probably without ubiquitination), leading to an intracellular accumulation of AZC. Interestingly, the rsp5A401E mutant also showed hypersensitivity to various stressors that induce protein misfolding: ethanol, heat shock, high temperature in a rich medium, toxic amino acid analogs, and oxidative treatments (Hoshikawa et al. 2003). Based on these findings, we speculated that Rsp5 is involved in a selective degradation of stress-induced abnormal proteins, and in a nitrogen-regulated degradation of Gap1. In general, the accumulation of stress-induced abnormal proteins poses a serious challenge for cells. Two strategies to address such an accumulation can be considered: degradation in the proteasome or vacuole, or refolding by molecular chaperones, such as stress proteins. It is of interest to determine whether or how Rsp5 is involved in these processes. We observed that when the rsp5A401E mutant was exposed to ethanol, a temperature up-shift, or sorbitol, significantly less transcription of stress protein genes occurred compared to that in the wild-type strain (Haitani, Shimoi and Takagi 2006). The amounts of the transcription factors Hsf1 and Msn4 in the rsp5A401E mutant were remarkably defective, suggesting that (i) expression of stress proteins are mediated by Rsp5 and (ii) Rsp5 primarily regulates the post-translational modification of Hsf1 and Msn4. After rsp5A401E cells were exposed to ethanol and a temperature upshift, the protein levels of Hsf1 and Msn2/4 were remarkably defective in the cells, although these proteins were localized mainly in the cell nucleus under these stress conditions (Haitani and Takagi 2008). The mRNAs of HSF1 and MSN2/4 also accumulated in the nuclei of rsp5A401E cells, indicating that Rsp5 may be required for the nuclear export of these mRNAs. Rsp5 primarily regulates the expressions of these 2 transcription factors at the post-transcriptional level and is involved in the repair of stress-induced abnormal proteins (Figure 9a). Figure 9. Open in new tabDownload slide Proposed models for Rsp5-mediated repair and degradation of stress-induced abnormal proteins in S. cerevisiae (Haitani et al. 2006; 2008; Hiraishi et al. 2009b). (a) Rsp5 is involved in the nuclear transport of the mRNAs of Hsf1 and Msn2, which are transcription factors of stress proteins that function as molecular chaperones. Under high-temperature and ethanol stress conditions, Rsp5 regulates the translation of Hsf1 and Msn2/4 via ubiquitination of the substrate protein (?) related to the nuclear transport of RNA molecules. (b) The α-subunit Egd2 of the nascent polypeptide-associated complex is ubiquitinated by Rsp5 and degraded under high-temperature and ethanol stress conditions. Although it has not been confirmed whether Egd2 becomes an abnormal protein, we found that Gap1 disappears from the plasma membrane in an Rsp5-dependent manner and is transported to the vacuole under ethanol stress conditions (Shiga et al. 2014). Figure 9. Open in new tabDownload slide Proposed models for Rsp5-mediated repair and degradation of stress-induced abnormal proteins in S. cerevisiae (Haitani et al. 2006; 2008; Hiraishi et al. 2009b). (a) Rsp5 is involved in the nuclear transport of the mRNAs of Hsf1 and Msn2, which are transcription factors of stress proteins that function as molecular chaperones. Under high-temperature and ethanol stress conditions, Rsp5 regulates the translation of Hsf1 and Msn2/4 via ubiquitination of the substrate protein (?) related to the nuclear transport of RNA molecules. (b) The α-subunit Egd2 of the nascent polypeptide-associated complex is ubiquitinated by Rsp5 and degraded under high-temperature and ethanol stress conditions. Although it has not been confirmed whether Egd2 becomes an abnormal protein, we found that Gap1 disappears from the plasma membrane in an Rsp5-dependent manner and is transported to the vacuole under ethanol stress conditions (Shiga et al. 2014). We conducted a comparative proteome analysis of the rsp5A401E mutant under stress conditions to identify the protein substrates of Rsp5, and we observed that several proteins, including the alpha subunit of nascent polypeptide-associated complex (αNAC; Egd2) were accumulated in this mutant (Hiraishi et al. 2009b). Interestingly, under the stress conditions used, Egd2 was ubiquitinated in the wild-type cells but not in the rsp5A401E mutant cells. At elevated temperature, we also detected the in vitro ubiquitination of Egd2 by Rsp5. The ubiquitination of Egd2 is independent of the RING-type Ub ligase Not4. Our study also demonstrated that under stress conditions, Egd2 was degraded mainly via the proteasome pathway, which strongly implies the involvement of Rsp5 in a selective ubiquitination and degradation of stress-induced unstable proteins, such as Egd2 (Figure 9b). We also investigated the intracellular trafficking of Gap1 as a model substrate of cell membrane proteins under various stress conditions in order to determine whether aberrant plasma membrane proteins are ubiquitinated by Rsp5 and whether they are eliminated from the plasma membrane via endocytosis (Shiga et al. 2014). When we exposed S. cerevisiae cells to a high concentration of ethanol, Gap1 was inactivated and ubiquitinated by Rsp5 for endocytosis. Other environmental stresses, such as H2O2, high temperature, and LiCl, also promoted the endocytosis of Gap1. Similar intracellular trafficking occurred in other plasma membrane proteins (Agp1, Tat2, and Gnp1) following the high-ethanol treatment, suggesting that in yeast, quality control induced by stress is a common process that requires Rsp5 for plasma membrane proteins (Figure 10a). Under the stress conditions, Gap1 responded to environmental changes, was recognized by various adapter proteins, and was ubiquitinated. These results indicated the existence of a specific protein quality-control mechanism in yeast under stress conditions. We also observed a new mechanism for the substrate specificity of Rsp5: regulation by the phosphorylation of conserved Thr residues in the three WW domains. These observations clarified aspects of the specificity and diversity of the recognition of substrates by Rsp5 (Sasaki and Takagi 2013; Watanabe et al. 2015). Figure 10. Open in new tabDownload slide Proposed models for Rsp5- and Ub system-mediated stress responses in S. cerevisiae. Detailed description of the model is referred to in the text. (a) The Gap1p endocytosis in response to environmental signals (Shiga et al. 2014). We discovered Bul1/2-dependent and independent endocytosis of plasma membrane proteins in response to various stresses, namely the plasma membrane quality control. (b) Proteasome gene expression as a freeze-thaw stress response in S. cerevisiae (Watanabe et al. 2018). Deletion of PDR1 or PDR3 impaired model dough fermentation after freezing preservation to the same extent as disruption of the RPN4 gene, suggesting the importance of individual transcription factors in freeze-thaw stress responses. Figure 10. Open in new tabDownload slide Proposed models for Rsp5- and Ub system-mediated stress responses in S. cerevisiae. Detailed description of the model is referred to in the text. (a) The Gap1p endocytosis in response to environmental signals (Shiga et al. 2014). We discovered Bul1/2-dependent and independent endocytosis of plasma membrane proteins in response to various stresses, namely the plasma membrane quality control. (b) Proteasome gene expression as a freeze-thaw stress response in S. cerevisiae (Watanabe et al. 2018). Deletion of PDR1 or PDR3 impaired model dough fermentation after freezing preservation to the same extent as disruption of the RPN4 gene, suggesting the importance of individual transcription factors in freeze-thaw stress responses. In another study from our research group, the Thr357Ala/Lys764Glu variant Rsp5 was demonstrated to induce the constitutive inactivation of Gap1 (Haitani et al. 2009). The Thr357Ala substitution in the WW2 domain constitutively caused a downregulation of 4 Pro permeases, leading to the AZC tolerance of yeast cells. Gap1 was highly ubiquitinated in RSP5T357A cells and continuously delivered to the vacuole from the Golgi apparatus without sorting to the plasma membrane. Our findings also indicated that Thr phosphorylation occurred in all three WW domains and, interestingly, that Thr357 in the WW2 domain of Rsp5 was phosphorylated, which is in agreement with our in vitro observation of the mouse Rsp5 orthologue (Sasaki and Takagi 2013). We studied the effects of substitutions of amino acids in individual WW domains on the endocytosis of Gap1 via an interaction with the Art proteins, ie Bul1 and Bul2, and we observed that the rsp5W257F/P260A, rsp5W359F/P362A, and rsp5W415F/P418A mutations increased the sensitivity to AZC, resulted in the defective endocytosis of Gap1, and impaired interactions with Bul1. Thus, the molecular recognition by each WW domain is responsible for the cooperative interaction with Bul1. Taken together, our findings illustrate the cooperative and specific roles of the WW domains of Rsp5 in the regulation of Bul1/2-mediated cellular events (Watanabe et al. 2015). Biotechnological applications of the ubiquitin system in yeast I have attempted to improve the stress tolerance of yeasts by enhancing the Ub system, focusing mainly on the molecular functions of Rsp5 (Ub system engineering) as applied research. In laboratory yeast strains, the overexpression of Rsp5 along with the overexpression of a Ub-binding enzyme (Ubc1-13) improved the yeasts’ tolerance to various types of stress (Hiraishi, Mochizuki and Takagi 2006; Hiraishi et al. 2009a). From a practical point of view, the creation of new industrial yeast strains that are tolerant to high ethanol concentrations during sake brewing and to ROS generated during the manufacture of frozen dough may be achieved by the overexpression of Rsp5 or Ubc6 and the co-overexpression of Rsp5 and Ubc9, Ubc11, or Ubc13. The overexpression of Ub-related enzymes may be a useful for the breeding of novel stress-tolerant strains. Two Rsp5 variants (Thr255Ala, Thr357Ala) obtained by our introduction of random mutagenesis into the WW domain exhibited enhanced tolerance to sodium acetate and AZC (Haitani et al. 2009; Watcharawipas, Watanabe and Takagi 2017). We constructed bottom-fermenting beer yeast strains in which the S. cerevisiae-RSP5 or Saccharomyces bayanus (SB)-RSP5 is highly expressed and used for high-gravity fermentation (Ogata et al. 2012). The SB-RSP5-overexpressing yeast showed a higher fermentation rate than its parent strain. In addition, a bioethanol yeast strain that expresses the Thr255Ala variant exhibited increased initial fermentation rates in the presence of sodium acetate (Watcharawipas, Watanabe and Takagi 2017). This mutation may thus contribute to improvements of the production of bioethanol from lignocellulosic biomass. Our research also revealed that the Ub-proteasome system contributes to the degradation of proteins that are denatured by freezing, enabling baker's yeast to maintain high fermentation even after freezing (Watanabe et al. 2018). A marked impairment of model dough fermentation using frozen cells occurred when we treated laboratory yeast strains with the proteasome inhibitor MG132 or deleted the respective transcriptional activator gene for the proteasome genes, ie RPN4, PDR1, or PDR3. The proteasomal degradation of freeze-thawing-damaged proteins may thus guarantee high cell viability and fermentation performance. The strain that was sensitive to freeze-thawing stress was heterozygous at the PDR3 locus, and we observed that one of the alleles had a dominant-negative phenotype of slow fermentation. The results of our investigations revealed that the proteasomal degradation of ubiquitinated proteins of S. cerevisiae is an essential process in this yeast's freeze-thawing stress response (Figure 10b). Several upstream transcriptional activator genes for the proteasome components are responsible for the quality of fermentation after freezing preservation. Our findings further clarify the relationship between the proteasomal functions in S. cerevisiae and freeze-thawing stress, and this knowledge may contribute to the design of new high-performance yeast products. We constructed a drug screening system that uses the stress sensitivity of an Rsp5 function-deficient strain (Ala401Glu) (Uesugi et al. 2014), and with it we obtained an interesting variant (Pro343Ser) that promotes the degradation of a small lipid-binding protein that is implicated in several neurodegenerative diseases, ie human α-synuclein (α-syn) (Wijayanti et al. 2015). To investigate the mechanism of the Rsp5-mediated detoxification of α-syn, we isolated 4 new Rsp5 variants (Thr255Ala, Asp295Gly, Pro343Ser, and Asn427Asp) that each conferred α-syn tolerance to yeast cells. These mutants were phenotypically distinct from our previously identified RSP5T357A mutation, which increases the ubiquitination of Gap1. Among these variants, the RSP5P343S substitution accelerated the degradation of α-syn, suppressed the accumulation of intracellular ROS, and enhanced the interaction with α-syn and its ubiquitination. These novel mutations might thus be useful for identifying the molecular basis on which disused proteins are specifically recognized and effectively removed, and for screening drug candidates for neurodegenerative diseases. Figure 11 summarizes the functions of Rsp5 variants with an amino acid change in the WW domains. Figure 11. Open in new tabDownload slide Molecular functions of Rsp5 variants with an amino acid change in the WW domains Thr255Ala (Watcharawipas et al. 2017), Pro343Ser (Wijayanti et al. 2015), Thr357Ala (Sasaki and Takagi 2013), and Ala401Glu (Shiga et al. 2014). We clarified a part of the specificity and diversity of substrate recognition by Rsp5. Detailed description of the model is referred to in the text. Figure 11. Open in new tabDownload slide Molecular functions of Rsp5 variants with an amino acid change in the WW domains Thr255Ala (Watcharawipas et al. 2017), Pro343Ser (Wijayanti et al. 2015), Thr357Ala (Sasaki and Takagi 2013), and Ala401Glu (Shiga et al. 2014). We clarified a part of the specificity and diversity of substrate recognition by Rsp5. Detailed description of the model is referred to in the text. The improvement of stress tolerance by the regulation of gene expression of transcription factors The functions of transcriptional factors are another focus of my research. I have attempted to enhance industrial yeasts’ stress tolerance. Heat-shock factor 1 (Hsf1) is a transcription activator that governs the expression of several heat-shock proteins in response to elevated temperature (Smith and Yaffe 1991). Under oxidative stress, the basic leucine-zipper transcription factor Yap1 is required for the induction of stress-responsive genes (Moye-Rowley, Harshman and Parker 1989). S. cerevisiae has also developed species-specific transcription factors that have pivotal roles in stress responses to diverse stress conditions by transcribing hundreds of stress-related genes: Msn2 and its partially redundant paralogue Msn4 (Msn2/4) (Martínez-Pastor et al. 1996; Berry and Gasch 2008). In a bioethanol yeast strain, the overexpression of the transcriptional activator Haa1, which is involved in weak acid stress response, in the presence of acetic acid used in the pretreatment of biomass enhanced the cell growth and increased the ethanol production (Inaba et al. 2013). Msn2/4 Msn2 and Msn4 have important roles in the mediation of a wide range of responses to temperature upshift, freezing stress, high ethanol concentrations, oxidative stress, osmotic shock, and glucose starvation (Izawa et al. 2007; Sadeh et al. 2011; Sasano et al. 2012b). Msn2 activates the transcription of DOT1, which encodes a repressor of ribosome biogenesis gene (Elfving et al. 2014). The transcription of XBP1, which encodes a repressor of cell cycle-associated genes, is also Msn2-dependent (Miles et al. 2013). In efforts to determine how Msn2 and Msn4 contribute to stress responses, comprehensive investigations of the downstream target genes of Msn2/4 have been conducted; their findings are as follows. (i) Msn2 and Msn4 directly induce the expression of the genes encoding antioxidant enzymes (eg catalase, thiol peroxidases, and superoxide dismutases) (Hasan et al. 2002; Drakulic et al. 2005; Sadeh et al. 2011). (ii) Msn2 and Msn4 activate the gene expressions for stress responses at the protein level. These 2 transcription factors are essential in the induction of genes for heat-shock proteins, including mainly molecular chaperons (Kandror et al. 2004). (iii) As a response to stress, Msn2 and Msn4 trigger metabolic reprogramming by inducing the expression of mitochondrial respiratory genes, glycogen synthetic genes, pentose phosphate pathway genes, and trehalose synthetic genes (Elfving et al. 2014). In efforts to improve the stress tolerance and the fermentation performance of industrial yeast strains, researchers have applied the overexpression of the MSN2 or MSN4 gene (Cardona et al. 2007; Watanabe et al. 2009). Baker's yeast that overexpressed MSN2 demonstrated higher tolerance to freezing stress and enhanced fermentation ability in frozen dough (Sasano et al. 2012b). In second-generation bioethanol production using lignocellulosic biomass, several growth/fermentation inhibitors (eg furfural and 5-hydroxymethylfurfural) are generated and then produce ROS (Allen et al. 2010); the overexpression of MSN2 in bioethanol yeast strains grown in the presence of furfural upregulated the transcription of antioxidant gene, which in turn increased the fermentation rate (Sasano et al. 2012e). Another bioethanol yeast strain that overexpresses MSN2 was able to grow well in the presence of sucrose, which mimics the highly osmotic environment of the biomass (Nur'utami et al. 2017). Our group recently demonstrated that MSN2 overexpression shortens the replicative lifespan of S. cerevisiae cells by reducing the intracellular Pro levels (Mukai et al. 2019). Compared to the wild-type cells, higher sensitivity to toxic amino acid analogues (eg AZC) was observed in MSN2-overexpressing cells (Nanyan et al. 2019). The intracellular content of AZC was increased by the overexpression of MSN2, suggesting that Msn2 positively regulates the uptake of Pro. MSN2 overexpression also markedly increased the amount of the amino acid permease Gnp1, which is involved in the toxicity of AZC, suggesting a novel link between the Msn2-mediated global stress response and the amino acid homeostasis in S. cerevisiae. We observed that MSN2 overexpression increased the levels of ubiquitinated protein with reduced free ubiquitin (Nanyan et al. 2020). We speculate that the Msn2-mediated uptake of amino acids may contribute to global stress responses in addition to the known effects of Msn2 (eg upregulating antioxidant enzyme genes, protein quality control via molecular chaperones, and reprogramming carbon metabolism). MSN2 overexpression and Pro accumulation can be expected to help improve the stress tolerance of industrial yeasts. The co-overexpression of MSN2 and UBP6 may increase tolerance of yeast cells to the proteotoxic stress caused by the intracellular accumulation of misfolded proteins (Nanyan and Takagi 2020). Pog1 We isolated the POG1 (promoter of growth) gene as the suppressor gene whose overexpression enables the rsp5A401E mutant to grow at high temperatures (Demae et al. 2007). The yeast cells that overexpress POG1 were more tolerant to LiCl treatment than cells harboring only the vector (Demae et al. 2007). The POG1 gene is not essential for cell growth, and it encodes a nuclear chromatin-associated protein that has no significant homology to any known protein. POG1 overexpression inhibits both the G1 arrest that is induced by α-factor, which synchronizes the cell cycle in the G1 phase, and the transcriptional repression of the cell cycle-related gene CLN2 (Leza and Elion 1999). Pog1 is likely to be a transcriptional activator that regulates genes involved in cell-cycle regulation, spindle assembly, and cytoskeletal function (Horak et al. 2002). POG1 overexpression also led to an increased transcriptional level of 2 stress protein genes (HSP12 and DDR2) and 2 proteasome component genes (RPT2 and RPN7), which indicates an enhancement of the pathways of protein refolding and degradation (Demae et al. 2007). One of our studies revealed that POG1 overexpression in a baker's yeast strain conferred increased fermentation ability in high-sucrose-containing dough used for sweet dough baking (Sasano et al. 2013). The deletion of POG1 greatly increased the fermentation ability in bread dough after freeze-thawing stress, which would be a useful characteristic for the baking of frozen dough. The engineering of yeast strains to control the expression of POG1 could thus become a novel method for the molecular breeding of baker's yeast (Sasano et al. 2013). With the use of yeast two-hybrid screening, we identified the RING-type Ub ligase Dma2, which is involved in cell-cycle regulation, as the protein interacting with Pog1 (Oshiro and Takagi et al. 2014). The interaction between Pog1 and Dma2 requires the phosphorylation of Thr253 in Pog1 and the Forkhead-associated domain in Dma2. We suspect that Pog1 is ubiquitinated by Dma2, but we observed that a dephosphorylation-mimic mutation in POG1 increased the level if Pog1, possibly due to the failure of ubiquitination. Furthermore, Pog1 might control the cell cycle, and its phosphorylated form may be downregulated by Dma2. Future perspectives and conclusions In addition to the above-described research, our research group has comprehensively analyzed the gene expression profiles of sake yeast and baker's yeast under several practical stress conditions, and we have accumulated findings that contribute to the breeding of industrial strains with enhanced stress tolerance and fermentation ability (Ando et al. 2006, 2007; Wu et al. 2006; Shima, Ando and Takagi 2008). S. cerevisiae as a suitable host for food- and pharmaceutical-grade products has been examined in many studies based on its status as a substance that the U.S. Food and Drug Administration (FDA) has designated “generally recognized as safe (GRAS).” Since S. cerevisiae is considered reliable and safe in food production, the development of novel S. cerevisiae strains with enhanced stress tolerance will provide significant contributions to food-related industries. Currently, the CRISPR/Cas9 system becomes popular for targeted genome editing. This system can be used for a number of yeast strain engineering to integrate multiple DNA constructs into targeted locations in the genome with a single transformation for rapid functional testing of combinations of mutations (Horwitz et al. 2015). In addition, SC yeast does not have to be treated the same as GM yeast. However, the food industry in Japan has not accepted the CRISPR/Cas9 and self-cloning technologies yet. I propose another option: a method of constructing commercial yeast strains that have the same stress tolerance mechanism as those described herein, and that consumers would consider acceptable. The method is based on the following knowledge (Figure 12). AZC causes a misfolding of the proteins into which it is incorporated competitively with Pro, thus inhibiting the cell growth. However, cells that accumulate large amounts of Pro, which dilutes the effect of AZC, are resistant to AZC. Two more mechanisms of resistance to AZC exist: (i) the cells detoxify AZC directly (eg via Mpr1-catalyzed acetylation) and (ii) cells do not incorporate AZC due to the degradation of Pro permeases or AZC-incorporated proteins mediated by Rsp5. Thus, with the introduction of a favorable mutation in the PRO1, MPR1, or RSP5 gene, yeast cells that accumulate large amounts of Pro, acetylate AZC efficiently, or degrade AZC-incorporated proteins are resistant to AZC. By applying treatment with the conventional mutagen, we will be able to obtain beneficial strains derived from AZC-resistant mutants, as some Pro1, Mpr1, or Rsp5 variants may confer stress tolerance to yeast cells. Figure 12. Open in new tabDownload slide Proposed method for the construction of yeast strains available in food industry. According to this method, we have isolated Pro-accumulating sake and baker's yeast strains (Tsolmonbaatar et al. 2016; Murakami et al. 2020). Detailed description of the model is referred to in the text. Figure 12. Open in new tabDownload slide Proposed method for the construction of yeast strains available in food industry. According to this method, we have isolated Pro-accumulating sake and baker's yeast strains (Tsolmonbaatar et al. 2016; Murakami et al. 2020). Detailed description of the model is referred to in the text. Over the past 25 years, exploring a new idea based on the metabolic regulation of amino acids and proteins, I have discovered several stress tolerance mechanisms of laboratory yeasts that differ from those derived from “conventional” yeast molecular genetics. By developing high functionality in industrial yeast, I have also applied the obtained knowledge to the breeding of practical strains. The results will contribute to both basic research on stress biology in higher organisms and prokaryotes and the improved production of high-value added chemicals by yeasts, bacteria, and plants. Acknowledgments The studies described in this review were conducted at the Department of Bioscience, Fukui Prefectural University (FPU), and at the Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), for the past 25 years. I am sincerely grateful to all of my colleagues (faculty staffs and undergraduates/graduate students) who worked with me or assisted my research, particularly Drs. Shigeru Nakamori (Emeritus Prof.), Masaru Wada, and Yoshimitsu Hamano in FPU and Drs. Nobuyuki Yoshida, Daisuke Watanabe, Ryo Nasuno, and Akira Nishimura in NAIST, for their valuable discussion and continuous encouragement. I thank all of my collaborators in the industry–academia–government field, particularly Drs. Takao Hibi (FPU), Toshio Hakoshima (NAIST), Ken-ichi Kimura (Iwate Univ.), Daisuke Hagiwara (Chiba Univ.), Yukio Mukai (Nagahama Inst. Bio-Sci. Tech.), Jun Shima (Natl. Food Res. Inst.), Hitoshi Shimoi (Natl. Res. Inst. Brew.), Yoshito Kubo (Fukui Pref. Food Process. Res. Inst.), and Masataka Ohashi (Nara Pref. Inst. Ind. Develop.) and private companies (Eisai, Shimadzu, Asahi Breweries, Oriental Yeast, TableMark, Gekkeikan, BioJet), for their fruitful joint research. I would also like to express my deep appreciation to Ajinomoto for guiding me to microbial research and for supporting me for a long time. Finally, I am thankful to Drs. Sakayu Shimizu (Emeritus Prof., Kyoto Univ.), Andriy Sibirny, and Charles Abbas, who are former Presidents of International Commission on Yeasts, and Masayori Inouye (Distinguished Prof., Robert Wood Johnson Med. Sch. at Rutgers Univ.) for their special encouragement. Funding The preparation of this review was supported by a Grant-in-Aid for Scientific Research (S) (19H05639) and a Grant-in-Aid for Challenging Exploratory Research (19K22282) from Japan Society for the Promotion of Science (JSPS) to H.T. and in part by a grant from the Project of the NARO Bio-oriented Technology Research Advancement Institution (Research program on development of innovative technology) (30017B) to H.T. Disclosure statement No potential conflict of interest was reported by the author. Data availability The data that support the findings of this study are openly available at https://doi.org/10.1093/bbb/zbab022. References Abe T , Toyokawa Y, Sugimoto Y et al. Characterization of a new Saccharomyces cerevisiae isolated from hibiscus flower and its mutant with l-leucine accumulation for awamori brewing . Front Genet 2019 ; 10 : 490 . Google Scholar Crossref Search ADS PubMed WorldCat Adak S , Aulak KS, Stuehr DJ. 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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.
TI - Molecular mechanisms and highly functional development for stress tolerance of the yeast Saccharomyces cerevisiae
JO - Bioscience Biotechnology and Biochemistry
DO - 10.1093/bbb/zbab022
DA - 2021-04-24
UR - https://www.deepdyve.com/lp/oxford-university-press/molecular-mechanisms-and-highly-functional-development-for-stress-p8fVLhOpLG
SP - 1017
EP - 1037
VL - 85
IS - 5
DP - DeepDyve
ER -